Antibodies capable of specifically binding two epitopes on tissue factor pathway inhibitor

ABSTRACT

The application discloses a combination of two monospecific TFPI antibodies, wherein one antibody is capable of specifically binding TFPI (1-181) and the other antibody is capable of specifically binding TFPI (182-276), as well as bispecific anti-TFPI antibodies derived from two such monospecific antibodies. Both the combination of the two monospecific antibodies and the bispecific antibody strongly enhance thrombin generation by neutralising full length TFPIα, even where the concentration of TFPI is abnormally elevated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/775,108, filed Sep. 11, 2015, which is a 35 U.S.C. § 371 NationalStage application of International Application PCT/EP2014/055055 (WO2014/140240), filed Mar. 14, 2014, which claims priority to EuropeanPatent Application 13159515.9, filed Mar. 15, 2013 and U.S. ProvisionalApplication 61/789,274, filed Mar. 15, 2013; the contents of which areincorporated herein by reference.

SEQUENCE LISTING

In accordance with 37 C.F.R. § 1.52(e)(5), Applicants enclose herewiththe Sequence Listing for the above-captioned application entitled8678US03_SeqList.txt, created on Jan. 3, 2018. The Sequence Listing ismade up of 112 kilobytes, and the information contained in the attached“SEQUENCE LISTING” is identical to the information in the specificationas originally filed. No new matter is added.

FIELD OF THE INVENTION

The present invention relates to antibodies, and compositions thereof,that are capable of binding to an epitope in the N-terminal region(residues 1-181) present in both tissue factor pathway inhibitor alpha(TFPIα) and TFPI beta (TFPIβ) and to another epitope within theC-terminal part (residues 182-276), present only in full-length TFPIα.The invention also relates to the pharmaceutical and therapeutic uses ofsuch antibodies.

BACKGROUND

In the bleeding individual, coagulation is initiated by the TissueFactor/Factor VIIa (TF/FVIIa) complex when extravascular TF is exposedto FVIIIa in the blood. TF/FVIIa complex formation leads to theactivation of Factor X (FX) to FXa which, together with activated FactorV (FVa), generates a limited amount of thrombin. Small amounts ofthrombin activate platelets which, in turn, results in the surfaceexposure of platelet phospholipids that supports the assembly andbinding of the tenase complex composed of activated Factor VIII (FVIIIa)and Factor IX (IXa). The tenase complex is a very efficient catalyst ofFX activation and FXa generated in this second step serves as the activeprotease in the FVa/FXa pro-thrombinase complex responsible for thefinal thrombin burst. Thrombin cleaves fibrinogen to generate fibrinmonomers, which polymerise to form a fibrin network which seals theleaking vessel and stops the bleeding. The rapid and extensive thrombinburst is a prerequisite for the formation of a solid and stable fibrinclot.

An inadequate propagation of FXa and thrombin generation caused by FVIIIor FIX deficiency is the reason underlying the bleeding diathesis inhaemophilia A and B patients, respectively. In people with haemophilia,FXa generation is primarily driven by the TF/FVIIa complex because FVIIIor FIX deficiency leads to rudimentary FXa generation by the tenasecomplex. TF/FVIIa-mediated activation of FX to FXa is, however,temporary because tissue factor pathway inhibitor (TFPI) inhibits FactorXa and the TF/FVIIa complex in an auto-regulatory loop. Feed-backinhibition leads to formation of a TF/FVIIIa/FXa/TFPI complex.Neutralizing TFPI inhibition prolongs TF/FVIIa-mediated activation of FXduring initiation of coagulation, and thereby it promotes haemostasis inpeople with haemophilia with an inadequate FXa generation caused byimpaired tenase activity due to e.g. FVIII or FIX deficiency.

TFPI is a slow tight-binding competitive inhibitor which regulates FXactivation and activity through inhibition of both TF-FVIIa and FXa.TFPI inhibition of FXa occurs in a biphasic reaction that initiallyleads to a loose TFPI-FXa complex which slowly rearranges to a tightbinding TFPI-FXa complex where the second Kunitz-type inhibitor domainof TFPI (KPI-2) binds and blocks the active site of FXa. Followinginitiation of coagulation, TF/FVIIa-mediated FXa generation is tightlydown-regulated by TFPI. TF/FVIIa is inhibited by TFPI in a process whichas a rate limiting step involves TFPI inhibition of FXa, either when FXais bound to the TF/FVIIa complex or bound in its near vicinity on themembrane (Baugh et al., 1998, JBC, 273: 4378-4386). The firstKunitz-type inhibitor domain of TFPI (KPI-1) contributes to theformation of the tight TFPI-FXa complex and it directly binds and blocksthe active site of TF-bound FVIIIa. The C-terminal part of TFPI,consisting of the third Kunitz-type inhibitor domain, KPI-3, and thebasic C-terminal tail, does not have any direct inhibitory activity, butit enhances the formation of the TFPI-FXa complex and it binds toProtein S and to heparin-like molecules which are involved in localisingTFPIα to the vascular surface.

TFPIα inhibits FXa-mediated thrombin generation by the prothrombinasecomplex at physiologically relevant TFPIα concentrations. This feed-backinhibition works primarily as a temporal impediment of the initiationphase of clot formation. TFPIα inhibition of FXa in the prothrombinasecomplex is mediated via a high-affinity interaction between the basicregion of TFPIα and an acidic region in FV, which is exposed on plateletFVa and on FVa when initially activated by FXa. The FVa-dependentinhibitory activity of TFPIα is lost upon removal of the acidic regionwhen FVa is further cleaved by thrombin generated by the prothrombinasecomplex. TFPIβ lacks the C-terminal basic region and is therefore not aninhibitor of prothrombinase activity (Wood et al. 2013, PNAS, 110:17838-843).

Antibodies that are capable of binding TFPI are known in the art. Forexample, WO2010/072691, WO2012/001087 and WO2012/135671 disclosemonoclonal antibodies (mAbs), each of which is capable of binding to onespecific epitope of TFPI. The following limitations may apply to such anantibody that targets a single TFPI epitope, e.g. on a KPI domain,typically restricted to the paratope area of an antibody defined by asingle variable region. First of all, the final inhibition of theTF/FVIIa/FXa complex is dependent on several interactions betweencomplementary areas scattered over TFPI and the TF/FVIIa/FXa complex.This applies not only to the direct binding of KPI-1 and KPI-2 of TFPIto the active sites of FVIIa and FXa, respectively, but also tointeractions with TF/FVIIa/FXa exosites which involve regions of the N-and C-terminal regions of TFPI. A monoclonal antibody that binds, forexample, a single KPI may not be capable of completely blocking allinhibitory functions of TFPI, particularly at physiologically elevatedconcentrations of TFPI. Secondly, targeting TFPI with a monoclonalantibody or fragment thereof may cause TFPI to accumulate in thecirculation as a result of a reduced renal clearance of the TFPI-mAbcomplex, or as a result of other clearance mechanisms which are reduceddue to TFPI-mAb complex formation. Dosing of some monoclonal antibodiesmay also cause release of TFPI from the endothelium and rapidlyincreasing plasma TFPI levels, similarly to what has been observed afterdosing of heparin or an aptamer, which binds to KPI-3 and the C-terminaltail of TFPI (Wong et al. Haemophilia, 2012, 18: LB-WE 03.1 p 831,Hoppensteadt et al., Thromb. Res., 77: 175-185). Thirdly, it may bedesirable to target a specific pool of TFPI. Full length circulatingTFPIα is thought to be of particular importance for the regulation ofcoagulation at a site of injury. The fact that, of all TFPI pools, onlyfull-length TFPIα possesses an exposed C-terminal region (residues182-276) makes it possible to selectively target the full-length TFPIαpool. By only targeting the full-length TFPIα pool and not, for example,TFPIβ or lipoprotein associated TFPI, target mediated drug dispositionmay be reduced, leading to prolonged in vivo drug half-lives and lowerdose requirements. However, known antibodies specifically targeting theC-terminal region of TFPIα are not capable of completely neutralisingTFPIα activity, especially at elevated concentrations of TFPI. Theinventors envisage that the antibodies—and combinations thereof—that aredisclosed herein may address such limitations.

SUMMARY

The invention relates to a bispecific antibody that is capable ofspecifically binding two epitopes on TFPI, the first of which is locatedwithin TFPI (1-181) (SEQ ID NO: 1) and the second of which is locatedwithin TFPI (182-276) (numbering relative to SEQ ID NO: 1). The first ofsaid epitopes may be on the KPI-1 domain of TFPI. Alternatively, thefirst of said epitopes may be on the KPI-2 domain of TFPI. The second ofsaid epitopes may be on the KPI-3 domain of TFPI. The bispecificantibody may be a full length antibody or it may be a conjugate orfusion protein of two antibody fragments, such as a Fab-Fab conjugate orfusion protein.

The invention also relates to a combination of two monospecificantibodies, wherein the first monospecific antibody is capable ofspecifically binding TFPI (1-181) and the second monospecific antibodyis capable of specifically binding TFPI (182-276). The first antibodymay specifically bind the KPI-1 domain of TFPI or the KPI-2 domain ofTFPI and the second antibody may specifically bind the KPI-3 domain ofTFPI.

The invention, moreover, relates to bispecific antibodies whichselectively target full-length TFPIα.

The invention also relates to a pharmaceutical formulation comprisingthe bispecific antibody or the combination of monospecific antibodiesaccording to the invention, any of which may find utility as amedicament.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that thrombin generation was strongly enhanced by combinedantibody targeting of KPI-2 and KPI-3 of TFPI in human plasma underhaemophilia A-like conditions with increased TFPI levels. Thrombingeneration (TGT assay) was measured with a pool of normal human plasma(NHP) with 10 μM phosphatidyl choline/phosphatidyl serine vesicles.Initiation of coagulation was induced by re-calcification and additionof 1 pM lipidated tissue factor (Innovin®). Curve (a) shows the resultobtained in NHP without further additions. Curve (b) shows the resultwhen haemophilia A-like conditions are obtained by the addition of 100μg/ml sheep anti human FVIII antibody (commercially available). Curve(c) shows that addition of 20 nM full-length TFPIα to haemophilia A-likeplasma completely suppressed thrombin generation. Curve (d) shows thatthe suppression of thrombin generation, as a result of 20 nM full-lengthTFPIα in combination with neutralisation of FVIII, was only partlyreversed by the addition of 200 nM mAb 2021 binding specifically toKPI-2. Curve (e) shows that these same conditions completely preventedthat 200 nM mAb 4F110 binding specifically to KPI-3 reversed TFPIinhibition such as to allow reversal of a detectable thrombingeneration. Curve (f) shows that, in contrast, a combination of 100 nMmAb 4F110 binding KPI-3 combined with 100 nM mAb 2021 binding KPI-2effectively reversed TFPI inhibition and restored thrombin generation toapproximately the level obtained in NHP without FVIII antibodies (curve(a)).

FIG. 2A-D shows the neutralisation by mAbs and Fab-Fab conjugates of theTFPIβ inhibition of TF/FVIIa-mediated FXa generation on the cellsurface. EA.hy926 wt cells were incubated with increasing concentrationsof mAbs or Fab-Fab conjugates. FXa activity in the supernatant wasmeasured after incubation with 50 pM FVIIa and 50 nM FX at 37° C. withvarious concentrations (0-60 nM) of mAb 2021 (A), mAb 4F110: (B),Fab-Fab conjugate 9002 (C), or Fab-Fab conjugate 9004 (D). Squares showthe effect of 0.5 mg/ml goat anti human TF polyclonal antibody togetherwith 60 nM mAb or Fab-Fab conjugate. Results are shown as mean±SD,(n=3).

FIG. 3 shows that thrombin generation was strongly enhanced by combinedantibody targeting of KPI-2 and KPI-3 of TFPI in human plasma underhaemophilia A-like conditions with increased TFPI levels using anasymmetric bispecific antibody format.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 represents the amino acid sequence of human TFPI alpha.

SEQ ID NO: 2 represents the amino acid sequence of TFPI (1-181).

SEQ ID NO: 3 represents the amino acid sequence of the heavy chain (HC)of Fab 0088.

SEQ ID NO: 4 represents the amino acid sequence of the light chain (LC)of Fab 0088.

SEQ ID NO: 5 represents the amino acid sequence of the heavy chain (HC)of Fab 0094.

SEQ ID NO: 6 represents the amino acid sequence of the light chain (LC)of Fab 0094.

SEQ ID NO: 7 represents the amino acid sequence of the heavy chain (HC)of Fab 0095.

SEQ ID NO: 8 represents the amino acid sequence of the light chain (LC)of Fab 0095.

SEQ ID NO: 9 represents the amino acid sequence of the heavy chain (HC)of Fab 0089

SEQ ID NO: 10 represents the amino acid sequence of the light chain (LC)of Fab 0089.

SEQ ID NO: 11 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 22F66.

SEQ ID NO: 12 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 22F66.

SEQ ID NO: 13 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 22F71.

SEQ ID NO: 14 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 22F71.

SEQ ID NO: 15 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 22F74.

SEQ ID NO: 16 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 22F74.

SEQ ID NOs: 17-21 represent the nucleotide sequences of primers.

SEQ ID NO: 22 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 1F91.

SEQ ID NO: 23 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 1F91.

SEQ ID NO: 24 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 2F3.

SEQ ID NO: 25 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 2F3.

SEQ ID NO: 26 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 2F22.

SEQ ID NO: 27 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 2F22.

SEQ ID NO: 28 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 2F35.

SEQ ID NO: 29 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 2F35.

SEQ ID NO: 30 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 2F45.

SEQ ID NO: 31 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 2F45.

SEQ ID NO: 32 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 2021

SEQ ID NO: 33 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 2021.

SEQ ID NO: 34 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 0094.

SEQ ID NO: 35 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 0094.

SEQ ID NO: 36 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 0095.

SEQ ID NO: 37 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 0095.

SEQ ID NO: 38 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 4F110.

SEQ ID NO: 39 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 4F110.

SEQ ID NO: 40 represents the amino acid sequence of the heavy chain (HC)of Fab 2F22.

SEQ ID NO: 41 represents the amino acid sequence of the light chain (LC)of Fab 2F22.

SEQ ID NO: 42 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 22F132.

SEQ ID NO: 43 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 22F132.

SEQ ID NO: 44 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 22F79.

SEQ ID NO: 45 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 2F79.

SEQ ID NO: 46 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 41F41.

SEQ ID NO: 47 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 41F41.

SEQ ID NO: 48 represents the amino acid sequence of the heavy chain (HC)of mAb 4F110.

SEQ ID NO: 49 represents the amino acid sequence of the light chain (LC)of mAb 4F110.

SEQ ID NO: 50 represents the amino acid sequence of the heavy chain (HC)of mAb 2021.

SEQ ID NO: 51 represents the amino acid sequence of the light chain (LC)of mAb 2021.

SEQ ID NO: 52 represents the amino acid sequence of the heavy chain (HC)of Fab 0296.

SEQ ID NO: 53 represents the amino acid sequence of the light chain (LC)of Fab 0296.

SEQ ID NO: 54 represents the amino acid sequence of the heavy chain (HC)of mAb 0094.

SEQ ID NO: 55 represents the amino acid sequence of the light chain (LC)of mAb 0094.

SEQ ID NO: 56 represents the amino acid sequence of the heavy chain (HC)of mAb 0095.

SEQ ID NO: 57 represents the amino acid sequence of the light chain (LC)of mAb 0095.

SEQ ID NO: 58 represents the amino acid sequence of the heavy chain (HC)of Fab 0313.

SEQ ID NO: 59 represents the amino acid sequence of the light chain (LC)of Fab 0313.

SEQ ID NO: 60 represents the amino acid sequence of the heavy chainvariable domain (VH) of mAb 41F30.

SEQ ID NO: 61 represents the amino acid sequence of the light chainvariable domain (VL) of mAb 41F30.

SEQ ID NO: 62 represents the amino acid sequence of tagged TFPIKPI-1/N-terminal

SEQ ID NO: 63 represents the amino acid sequence of the heavy chain (HC)of mAb 0309

SEQ ID NO: 64 represents the amino acid sequence of the light chain (LC)of mAb 0309

SEQ ID NO: 65 represents the amino acid sequence of the heavy chain(HC)) of mAb 0312.

SEQ ID NO: 66 represents the amino acid sequence of the light chain (LC)of mAb 0312.

SEQ ID NO: 67 represents the amino acid sequence of the heavy chain 1(HC1) of mAb 0325.

SEQ ID NO: 68 represents the amino acid sequence of the light chain 1(LC1) of mAb 0325

SEQ ID NO: 69 represents the amino acid sequence of the heavy chain 2(HC2) of mAb 0325.

SEQ ID NO: 70 represents the amino acid sequence of the light chain 2(LC2) of mAb 0325.

DESCRIPTION

In vivo, tissue factor pathway inhibitor (TFPI) is found in severalcompartments. A major fraction of TFPI is associated with the vascularendothelium and a minor fraction circulates in the blood. Two splicevariants of TFPI, TFPI alpha (TFPIα) and TFPI beta (TFPIβ) have beendescribed in humans. TFPIβ is presumably the predominant form expressedon the endothelial cell surface, whereas intracellular stores of TFPIαcan be released into the circulation upon certain stimuli. TFPIαcirculates in the blood as either full-length or truncated protein,associated with lipoproteins or in platelets.

Mature human TFPIα is a 276 amino acid protein (SEQ ID NO: 1) iscomposed of an acidic N-terminal region, three tandemly arrangedKunitz-type inhibitor domains (KPI-1, KPI-2 and KPI-3) interspersed bylinker regions and a basic C-terminal tail. The KPI-1, KPI-2 and KPI-3domains are defined as residues 26-76, residues 97-147 and residues189-239, respectively, of SEQ ID NO: 1. Mature TFPIβ is a 193 amino acidprotein covalently attached to the endothelial cell surface via aglycosylphosphatidylinositol (GPI)-anchor. The first 181 amino acids ofTFPIβ are identical to TFPIα (corresponding to residues 1-181 of SEQ IDNO: 1) whereas the final 12 amino acid of the C-terminal sequence isunrelated to TFPIα and has a GPI-anchor attached to residue 193.

The present invention relates to a combination of two monospecificantibodies that bind two distinct and/or unique epitopes on TFPIα. Theinvention also relates to a bispecific antibody that is derived from twosuch monospecific antibodies. One of the monospecific antibodies, fromwhich one antigen recognition site (or “arm”) of the bispecific antibodyis derived, is directed toward an epitope in the KPI-1/KPI-2 region ofTFPI, herein defined as TFPI (1-181), which may be human TFPI (1-181)(SEQ ID NO: 2). The second monospecific antibody, from which the secondantigen recognition site (or “arm”) of the bispecific antibody isderived, is directed toward the “KPI-3 region” only present in TFPIα,herein defined as TFPI (182-276).

“KPI-1/KPI-2 region” herein refers to amino acid residues 1-181 of TFPI(i.e. TFPI (1-181)), which may be human TFPI (1-181) (SEQ ID NO: 2) andtherefore encompasses the KPI-1 and KPI-2 domains of TFPI. “KPI-3region” herein refers to amino acid residues 182-276 of TFPI (i.e. TFPI(182-276)) and therefore encompasses the KPI-3 domain and the C-terminaltail of TFPI. Hence, one monospecific antibody or one arm of thebispecific antibody or antibody fragment is capable of specificallybinding to an epitope that is located within the KPI-1/KPI-2 region ofTFPI (i.e. TFPI 1-181), including the acidic region near the N-terminal,KPI-1, KPI-2, the linker region between KPI-1 and KPI-2 and the firstportion of the linker region between KPI-2 and KPI-3 of TFPI. The secondmonospecific antibody or second arm of the bispecific antibody iscapable of specifically binding to an epitope that is located withinamino acid residues 182-276 present only in full-length TFPIα, but notin TFPIβ. Residues 182-276 correspond to the second portion of thelinker region between KPI-2 and KPI-3, the KPI-3 domain and the basicC-terminal tail.

The combination of the two monospecific antibodies of the invention, orthe bispecific antibody of the invention, may have a superior profilewhen its pro-coagulant effect is compared to the anticipated additiveeffect of the individual antibodies from which it derives. For example,the two monospecific antibodies combined, or the bispecific antibody,may be capable of blocking all inhibitory functions of TFPIα, even whenthe concentration of TFPIα is elevated relative to, e.g. normalphysiological levels. Linkage of two antigen binding moieties directedtowards two separate epitopes of the invention, to obtain a bi-specificantibody or a Fab-Fab conjugate may, due to an avidity effect, lead to amore potent neutralisation of the TFPIα activity than that obtained bythe combined effect of the two separate antigen binding moieties. It isnot a prerequisite that both of the monospecific antibodies, from whichthe inventive combination or the bispecific antibody of the invention isderived, have a detectable pro-coagulant activity when tested alone, afinding which is particularly surprising. The effect arising from theantibody or the one arm of the bispecific antibody, directed toward theKPI-1/KPI-2 region, may modulate the activity of all pools of TFPIwhereas the superior effect of the combination of the two monospecificantibodies, or the bispecific antibody, will preferentially modulate theactivity of full length TFPIα.

A bispecific antibody of the invention may be capable of selectivelymodulating the activity of one specific pool of TFPI, namely TFPIα. Asingle arm of the bispecific antibody, capable of binding theKPI-1/KPI-2 region, may in itself, like a monospecific antibody, beincapable of significantly preventing the inhibitory activity of TFPI.It may, however, contribute more significantly to specific blockage ofTFPIα inhibition when the second arm of the bispecific antibody is boundto the KPI-3 region of TFPIα. Such a bispecific antibody may thereby becapable of neutralising the inhibition of full-length TFPIα. The bindingto TFPIβ of the said bispecific antibody will exclusively depend on thearm that is targeting the KPI-1/KPI-2 region. Weakening of the bindingto the KPI-1/KPI-2 region will preferentially decrease the binding toTFPIβ whereas neutralization of TFPIα inhibition still remainsessentially intact due to the targeting of the second arm of thebispecific antibody to the KPI-3 region. Due to reduced or absenttargeting to TFPIβ or lipoprotein associated TFPI, such weakening of theaffinity to the KPI-1/KPI-2 region of one arm in the said bispecificantibody may also result in reduced target mediated drug dispositionleading to a prolonged in vivo drug half-life and/or lower antibody doserequirements.

The affinity of the KPI-3 region targeting component of the bispecificantibody may alone be sufficient to ensure efficient engagement of TFPI.Due to the avidity effect it may then be possible to substantiallydiminish binding to TFPIβ on endothelial cells while still preserving anefficient effect in plasma with neutralization of TFPIα activity. Thismay be obtained by weakening of the binding of the KPI-1/KPI-2 targetingcomponent of the bispecific antibody to a point where the avidity effectof the two paratopes linked together is still present. Such antibodiesmay be constructed by selecting antibodies targeting certain epitopesand by tuning the absolute and relative affinities of the KPI-1/KPI-2and KPI-3 binding components of the bispecific antibody by methods knownto a person skilled in the art. Additionally, the selectivity and otherproperties of such a bispecific antibody may be modulated by varying thebispecific format.

The term “TFPI” as used herein encompasses naturally occurring forms oftissue factor pathway inhibitor (TFPI) that may be derived from anysuitable organism. For example, TFPI for use as described herein may bea mammalian TFPI, such as human, mouse, rat, primate, bovine, ovine,rabbit or porcine TFPI. Preferably the TFPI is human TFPI. The TFPI maybe a mature form of TFPI such as a TFPI protein that has undergonepost-translational processing within a suitable cell. Such a mature TFPIprotein may, for example, be glycosylated. The TFPI may be a full lengthTFPI protein. The term TFPI also encompasses variants, isoforms andother homologs of such TFPI molecules. TFPI activity refers to itsinhibitory activity. Variant TFPI molecules will generally becharacterised by having the same type of activity as naturally occurringTFPI, such as the ability to neutralise the catalytic activity of FXa,or the ability to inhibit a complex of TF-FVIIa/FXa.

The term “antibody” herein refers to a protein, derived from animmunoglobulin sequence, which is capable of specifically binding to anantigen or a portion thereof. The term antibody includes, but is notlimited to, full length antibodies of any class (or isotype), that is,IgA, IgD, IgE, IgG, IgM and/or IgY. The term may also include one ormore antigen-binding fragments of full length antibodies. An antibodythat specifically binds to an antigen, or portion thereof, may bindexclusively to that antigen, or a portion thereof, or it may bind to alimited number of homologous antigens, or portions thereof.

Natural full-length antibodies usually comprise at least fourpolypeptide chains: two heavy (H) chains and two light (L) chains thatare connected by disulfide bonds. In some cases, natural antibodiescomprise less than four chains, as in the case of the heavy chain onlyantibodies found in camelids (V_(H)H fragments) and the IgNARs found inChondrichthyes. One class of immunoglobulins of particularpharmaceutical interest is the IgGs. In humans, the IgG class may besub-divided into four sub-classes IgG1, IgG2, IgG3 and IgG4, based onthe sequence of their heavy chain constant regions. The light chains canbe divided into two types, kappa and lambda chains based on differencesin their sequence composition. IgG molecules are composed of two heavychains, interlinked by two or more disulfide bonds, and two lightchains, each attached to a heavy chain by a disulfide bond. An IgG heavychain may comprise a heavy chain variable region (VH) and up to threeheavy chain constant (CH) regions: CH1, CH2 and CH3. A light chain maycomprise a light chain variable region (VL) and a light chain constantregion (CL). VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs) orhypervariable regions (HvRs), interspersed with regions that are moreconserved, termed framework regions (FR). VH and VL regions aretypically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable domains with the hypervariableregions of the heavy and light chains form a domain that is capable ofinteracting with an antigen, whilst the constant region of an antibodymay mediate binding of the immunoglobulin to host tissues or factors,including, but not limited to various cells of the immune system(effector cells), Fc receptors and the first component (C1q) of the C1complex of the classical complement system.

Antibodies of the current invention may be isolated. The term “isolatedantibody” refers to an antibody that has been separated and/or recoveredfrom (an)other component(s) in the environment in which it was producedand/or that has been purified from a mixture of components present inthe environment in which it was produced.

The term “monospecific antibody”, as used herein, refers to an antibodythat has a single antigen recognition site (i.e. monovalent) or twoidentical antigen recognition sites (i.e. bivalent), each of which arespecific for one common target antigen.

Monospecific antibodies of the invention may be monoclonal antibodies,in the sense that they represent a set of unique heavy and light chainvariable domain sequences as expressed from a single B-cell or by aclonal population of B cells. Antibodies of the invention may beproduced and purified using various methods that are known to the personskilled in the art. For example, antibodies may be produced fromhybridoma cells. Antibodies may be produced by B-cell expansion.Antibodies or fragments thereof may be recombinantly expressed inmammalian or microbial expression systems, or by in vitro translation.Antibodies or fragments thereof may also be recombinantly expressed ascell surface bound molecules, by means of e.g. phage display, bacterialdisplay, yeast display, mammalian cell display or ribosome or mRNAdisplay. Once produced, antibodies may be screened for their ability tobind TFPI (1-181), full length TFPIα and TFPIβ, such as human TFPI(1-181), full length human TFPIα and human TFPIβ.

Various antigen-binding fragments of antibodies may also be monospecificantibodies according to the current invention, as it has been shown thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody. The term “antigen-binding fragment”of an antibody refers to one or more fragment(s) of an antibody thatretain(s) the ability to specifically bind to or recognise an antigen,such as TFPIα, such as human TFPIα (SEQ ID NO: 1), such as human TFPI(1-181) (SEQ ID NO:2), such as human TFPI (182-276, numbering accordingto SEQ ID NO: 1), such as TFPIβ, such as human TFPIβ or another targetmolecule, as described herein. Examples of antigen-binding fragmentsinclude Fab, Fab′, Fab₂, Fab′₂, FabS, Fv (typically the VL and VHdomains of a single arm of an antibody), single-chain Fv (scFv; see e.g.Bird et al., Science (1988) 242:42S-426; and Huston et al. PNAS (1988)85: 5879-5883), dsFv, Fd (typically the VH and CH1 domain), and dAb(typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains;monovalent molecules comprising a single VH and a single VL chain;minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see,e.g., III et al. Protein Eng (1997) 10:949-57); camel IgG; IgNAR; aswell as one or more isolated CDRs or a functional paratope, where theisolated CDRs or antigen-binding residues or polypeptides can beassociated or linked together so as to form a functional antibodyfragment. Various types of antibody fragments have been described orreviewed in, e.g., Holliger and Hudson, Nat Biotechnol (2005)23:1126-1136; WO2005040219, and published U.S. Patent Applications20050238646 and 20020161201. These antibody fragments may be obtainedusing conventional techniques known to those of skill in the art, andthe fragments may be screened for utility in the same manner as intactantibodies.

“Fab fragments” of an antibody, including “Fab”, “Fab′”, and “Fab′₂”fragments, can be derived from said antibody by cleavage of the heavychain in the hinge region on the N-terminal or C-terminal side of thehinge cysteine residues connecting the heavy chains of the antibody. A“Fab” fragment includes the variable and constant domains of the lightchain and the variable domain and the first constant domain (CH1) of theheavy chain. “Fab′₂” fragments comprise a pair of “Fab′” fragments thatare generally covalently linked by their hinge cysteines. A Fab′ isformally derived from a Fab′₂ fragment by cleavage of the hingedisulfide bonds connecting the heavy chains in the Fab′₂. Other chemicalcouplings than disulfide linkages of antibody fragments are also knownin the art. A Fab fragment retains the ability of the parent antibody tobind to its antigen, potentially with a lower affinity. Fab′₂ fragmentsare capable of divalent binding, whereas Fab and Fab′ fragments can bindmonovalently. Generally, Fab fragments lack the constant CH2 and CH3domains, i.e. the Fc part, where interaction with the Fc receptors wouldoccur. Thus, Fab fragments are in general devoid of effector functions.Fab fragments may be produced by methods known in the art, either byenzymatic cleavage of an antibody, e.g. using papain to obtain the Fabor pepsin to obtain the Fab′₂, Fab fragments including Fab, Fab′, Fab′₂may be produced recombinantly using techniques that are well known tothe person skilled in the art.

An “Fv” fragment is an antibody fragment that contains a completeantigen recognition and binding site, and generally comprises a dimer ofone heavy and one light chain variable domain in association that can becovalent in nature, for example in a single chain variable domainfragment (scFv). It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions or a subset thereof confer antigen-bindingspecificity to the antibody. However, even a single variable domaincomprising only three hypervariable regions specific for an antigen canretain the ability to recognise and bind antigen, although usually at alower affinity than the entire binding site (Cai & Garen, Proc. Natl.Acad. Sci. USA (1996) 93: 6280-6285). For example, naturally occurringcamelid antibodies that only have a heavy chain variable domain (VHH)can bind antigen (Desmyter et al., J. Biol. Chem. (2002) 277:23645-23650; Bond et al., J. Mol. Biol. (2003) 332: 643-655).

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, where these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains that enables the scFvto form the desired structure for antigen-binding. For a review of scFv,see Pluckthun, 1994, In: The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, in which fragments comprise a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain (VH and VL). By using a linker that is tooshort to allow pairing between the two variable domains on the samechain, the variable domains are forced to pair with complementarydomains of another chain, creating two antigen-binding sites. Diabodiesare described more fully, for example, in EP 404,097; WO 93/11161; andHollinger et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6444-6448.

The expression “linear antibodies” refers to antibodies as described inZapata et al., 1995, Protein Eng., 8(10):1057-1062. Briefly, theseantibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that,together with complementary light chain polypeptides, form a pair ofantigen-binding regions. Linear antibodies can be bispecific ormonospecific.

The term “monobody” as used herein, refers to an antigen-bindingmolecule with a heavy chain variable domain and no light chain variabledomain. A monobody can bind to an antigen in the absence of light chainsand typically has three hypervariable regions, for example CDRsdesignated CDRH1, CDRH2, and CDRH3. A heavy chain IgG monobody has twoheavy chain antigen-binding molecules connected by a disulfide bond. Theheavy chain variable domain comprises one or more hypervariable regions,preferably a CDRH3 or HVL-H3 region.

Antibody fragments may be obtained using conventional recombinant orprotein engineering techniques and the fragments can be screened forbinding to TFPI (1-181), full length TFPIα and TFPIβ, or anotherfunction, in the same manner as intact antibodies.

Antibody fragments of the invention may be made by truncation, e.g. byremoval of one or more amino acids from the N and/or C-terminal ends ofa polypeptide. Fragments may also be generated by one or more internaldeletions.

The term “bispecific antibody” herein refers to an antibody that has twodistinct and/or unique antigen recognition sites, or “arms”, whichenables it to engage two different antigens or two different epitopes onthe same antigen. The term “multispecific antibody” refers to anantibody with the ability to engage two or more different antigens ortwo or more different epitopes on the same antigen. Multispecificantibodies thus comprise bispecific antibodies.

Bispecific antibodies in full length IgG format, mimicking naturalantibodies, can be generated by fusion of two individual hybridomas toform a hybrid quadroma which produces a mixture of antibodies includinga fraction of bispecific heterodimerising antibodies (Chelius D. et al.;MAbs. 2010 May-June; 2(3): 309-319). Bispecific heterodimerisingantibodies may alternatively be produced by using recombinanttechnologies. Heterodimerisation can be also be achieved by engineeringthe dimerisation interface of the Fc region to promoteheterodimerisation. One example hereof are the so-called knob-in-holemutations where stearically bulky side chains (knobs) are introduced inone Fc matched by stearically small side chains (holes) on the oppositeFc thereby creating steric complementarity promoting heterodimerisation.Other methods for engineered heterodimerisation Fc interfaces areelectrostatic complementarity, fusion to non-IgG heterodimerisationdomains or utilising the natural Fab-arm exchange phenomenon of humanIgG4 to control heterodimerisation. Examples of heterodimerisedbispecific antibodies are well described in the literature, e.g. (KleinC, et al.; MAbs. 2012 November-December; 4(6): 653-663). Specialattention has to be paid to the light chains in heterodimericantibodies. Correct pairing of LCs and HCs can be accomplished by theuse of a common light chain. Again engineering of the LC/HC interfacecan be used to promote heterodimerisation or light chain cross-overengineering as in CrossMabs. In vitro re-assembly under mildly reducingconditions of antibodies from two individual IgGs containing appropriatemutations can also be used to generate bispecifics (e.g. Labrijn et al.,PNAS, 110, 5145-5150 (2013)). Also the natural Fab-arm exchange methodis reported to ensure correct light chains paring.

Multispecific antibody-based molecules may also be expressedrecombinantly as fusion proteins combining the natural modules of IgGsto form multispecific and multivalent antibody derivatives as describedin the literature. Examples of fusion antibodies are DVD-Igs, IgG-scFV,Diabodies, DARTs etc (Kontermann, MAbs. 2012 March-April 4(2): 182-197).Specific detection or purification tags, half-life extension moieties orother components can be incorporated in the fusion proteins. Additionalnon-IgG modalities may also be incorporated in the fusion proteins.Bispecific full length antibodies based on FC heterodimerisation arecommonly referred to as asymmetic IgGs, irrespective of the LC paringmethodology.

Multispecific antibody-based molecules may also be produces by chemicalconjugation or coupling of individual full length IgGs or coupling offragments of IgGs to form multispecific and multivalent antibodyderivatives as described in the literature. Examples of fusionantibodies are chemical coupled Fab′₂, IgG-dimer etc. (Kontermann, MAbs.2012 Mar.-Apr. 4(2): 182-197). Specific detection or purification tags,half-life extension molecules or other components can be incorporated inthe conjugate proteins. Additional non-IgG polypeptide may also beincorporated in the fusion proteins. An example of such a bispecificantibody is provided in the examples.

Multispecific molecules may also be produced by combining recombinantand chemical methods including those described above.

A bispecific antibody of the current invention may comprise anantigen-binding fragment of the mAb 2021 antibody, a monoclonal antibodythat was first described in WO2010/072691, which is hereby incorporatedby reference.

A bispecific antibody of the current invention may comprise anantigen-binding fragment of the mAb 2F22 antibody.

A bispecific antibody of the current invention may comprise anantigen-binding fragment of the mAb 2F3 antibody, the mAb 2F45 antibody,the mAb 1F91 antibody or the 2F35 antibody.

A bispecific antibody of the invention may further comprise anantigen-binding fragment of the mAb 4F110 antibody, the mAb 22F66antibody or the mAb 22F71 antibody, monoclonal antibodies which werefirst described in WO2012/001087; which is hereby incorporated byreference; or, alternatively, an antigen-binding fragment of the mAb22F74 antibody, as disclosed herein. For example, a bispecific antibodyof the invention may comprise a Fab fragment of one of theabove-mentioned antibodies. A bispecific antibody of the invention mayalso comprise a variant Fab fragment based on the one of theabove-mentioned antibodies, such as Fab 0094 or Fab 0095 or Fab 0313(derivatives from mAb 2021) as disclosed herein.

Bispecific, or bifunctional, antibodies of the invention can be obtainedby chemical conjugation of two antibodies or fragments thereof that bindto different epitopes of TFPI:

mAb1 (or fragment)-mAb2 (or fragment)

mAb1 (or fragment)-linker-mAb2 (or fragment)

The linkage can be a single covalent bond (direct linkage) or comprise abiradical generally described as:

wherein X is at least, but not limited to, one atom selected from thegroup of carbon, oxygen, sulfur, phosphor, and nitrogen. * shows thepositions of connections of this biradical. The term “biradical” refersto an even-electron chemical compound with two free radical centerswhich act independently of one another.

In one embodiment the linker is a chain composed of no more than 40atoms.

In one embodiment, a chemical moiety used in the linker comprises thebiradical with the structure

In one embodiment, the linker part comprises the biradical which has asymmetrical structure (homo-bifunctionalised liker).

The linker may also be a polymer of a structure which is similar to thedescription above.

In an embodiment, a chemical moiety used in the linker comprises apolymer: a macromolecule composed of two or more repeating structuralunits that are connected by covalent chemical bonds. Such a polymer maybe hydrophilic.

The term hydrophilic or “water-soluble” refers to moieties that havesome detectable degree of solubility in water. Methods to detect and/orquantify water solubility are well known in the art.

Exemplary water-soluble polymers according to the invention includepeptides, saccharides, (poly)ethers, (poly)amines, (poly)carboxylicacids and the like. Peptides can have mixed sequences or can be composedof a single amino acid, e.g., (poly)lysine. An exemplary polysaccharideis (poly)sialic acid. An exemplary (poly)ether is (poly)ethylene glycol.(Poly)ethylene imine is an exemplary polyamine, and (poly)acrylic acidis a representative (poly)carboxylic acid.

Many other polymers are also suitable for the invention. Polymerbackbones that are water-soluble are particularly useful in theinvention. Examples of suitable polymers include, but are not limitedto, other poly(alkylene glycols), such as poly(propylene glycol)(“PPG”), copolymers of ethylene glycol and propylene glycol and thelike, poly(oxyethylated polyol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly([alpha]-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety, aswell as copolymers, terpolymers, and mixtures thereof.

The polymeric linker is, preferably, linear.

Although the molecular weight of each individual polymer chain may vary,the average molecular weight of the polymer is typically in the range offrom about 1000 Da (1 kDa) to about 40,000 Da (40 kDa), such as about1000 Da to about 12,000 Da such as about 2,000 Da to about 11,000 Da,such as about 2000 Da to about 3,000 Da; about 3000 Da to about 4,000Da; about 4000 to about 5,000 Da; about 5000 to about 6,000 Da; about6,000 to about 7,000 Da; about 7,000 to about 8,000 Da; about 8,000 toabout 9,000 Da; about 9,000 to about 10,000 Da; or about 10,000 to about11,000 Da. It should be understood that these sizes represent estimatesrather than exact measures. According to a preferred embodiment, themolecules according to the invention are conjugated with a heterogeneouspopulation of hydrophilic polymers.

In a particular embodiment, a chemical moiety used in the linkercomprises polyethylene glycol (PEG).

The term “PEG” herein refers to a biradical comprising the structure

wherein n′ is an integer larger than 1.

PEG is prepared by polymerisation of ethylene oxide and is commerciallyavailable over a wide range of molecular weights. The PEG for useaccording to the present invention is, preferably, linear.

Furthermore, “PEG” may refer to a polyethylene glycol compound, orderivative thereof, with or without coupling agents, coupling oractivating moieties (e.g., with carboxylic acid/active ester, keto,alkoxyamine, thiol, triflate, tresylate, aziridine, oxirane, alkyne,azide or a maleimide moiety). The other linkers mentioned herein mayalso be with or without coupling agents, coupling or activating moieties(e.g., with carboxylic acid/active ester, keto, alkoxyamine, thiol,triflate, tresylate, aziridine, oxirane, alkyne, azide or a maleimidemoiety)

In one particular embodiment the PEG for use according to the inventionis monodisperse. In another particular embodiment, the PEG for useaccording to the invention is polydisperse.

Polydisperse PEG is composed of PEG molecules that have variousmolecular weights. The size distribution can be characterisedstatistically by its weight average molecular weight (Mw) and its numberaverage molecular weight (Mn), the ratio of which is called thepolydispersity index (Mw/Mn) (see e.g. “Polymer Synthesis andCharacterization”, J. A. Nairn, University of Utah, 2003). Mw and Mn canbe measured by mass spectroscopy.

The polydispersity index may be a number that is greater than or equalto one and it may be estimated from Gel Permeation Chromatographic data.When the polydispersity index is 1, the product is monodisperse and isthus made up of compounds with a single molecular weight. When thepolydispersity index is greater than 1 the polymer is polydisperse, andthe polydispersity index tells how broad the distribution of polymerswith different molecular weights is. The polydispersity index typicallyincreases with the molecular weight of the PEG. In particularembodiments, the polydispersity index of the PEG for use according tothe invention is i) below 1.06, ii) below 1.05, iii) below 1.04, iv)below 1.03 or v) between 1.02 and 1.03.

Different forms of PEG are available, depending on the initiator usedfor the polymerisation process.

Numerous methods for conjugation of PEG substituents are described inAdvanced Drug Delivery Reviews, 2002, 54, 459-476, Nature Reviews DrugDiscovery, 2003, 2, 214-221 DOI:10.1038/nrd1033, Adv Polym Sci, 2006,192, 95-134, DOI 10.1007/12_022, Springer-Verlag, Berlin Heidelberg,2005, and references therein. Alternatively, conjugation of thehydrophilic polymer substituent could take place by use of enzymaticmethods. Such methods are for instance use of transglutaminases asdescribed in WO2006134148.

To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the end groups of the polymer molecule are provided inactivated form, i.e. with reactive functional groups. Suitable activatedpolymer molecules are commercially available, e.g. from Sigma-AldrichCorporation, St. Louis, Mo., USA, Rapp Polymere GmbH, Tübingen, Germany,or from PolyMASC Pharmaceuticals plc, UK. Alternatively, the polymermolecules can be activated by conventional methods known in the art,e.g. as disclosed in WO 90/13540. Specific examples of activated PEGpolymers are disclosed in U.S. Pat. Nos. 5,932,462 and 5,643,575.Furthermore, the following publications disclose useful polymermolecules and/or PEGylation chemistries: WO2003/031464, WO2004/099231.

The conjugation of the monoclonal antibody, or fragment thereof, withthe activated polymer molecules may be conducted by use of anyconventional method, e.g. as described in the following references(which also describe suitable methods for activation of polymermolecules): R. F. Taylor, (1991), “Protein immobilisation. Fundamentaland applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”,Academic Press, N.Y., Bioconjugate Techniques, Second Edition, Greg T.Hermanson, 2008, Amsterdam, Elsevier). The skilled person would be awarethat the activation method and/or conjugation chemistry to be useddepends on the attachment group(s) of the polypeptide (examples of whichare given further above), as well as the functional groups of thepolymer (e.g. being amine, hydroxyl, carboxyl, aldehyde, sulfhydryl,succinimidyl, maleimide, vinylsulfone or haloacetate). The PEGylationmay be directed towards conjugation to available attachment groups onthe polypeptide (i.e. such attachment groups that are exposed at thesurface of the polypeptide) or may be directed towards one or morespecific attachment groups, e.g. the N-terminal amino group or a thiol.Furthermore, the conjugation may be achieved in one step or in astepwise manner.

In another embodiment, a chemical moiety used as the linker ishydroxyethyl starch. The term “hydroxyethyl starch” (HES/HAES), as usedherein, refers to a nonionic starch derivative. Different types ofhydroxyethyl starches are typically described by their average molecularweight, typically around 130 to 200 kDa.

In another embodiment, a chemical moiety used in the linker comprisespolysialic acid.

In another embodiment, a chemical moiety used in the linker comprisesheparosan polymer which is described in for instance Glycobiology (2011)21: 1331-1340.

In another embodiment, a chemical moiety in the linker is used to attachat least one of the proteins to a glycan: a polysaccharide or anoligosaccharide that is attached to a protein.

In another embodiment, a chemical moiety in the linker is used to attachat least one of the proteins to an O-linked glycan.

In another embodiment, a chemical moiety in the linker is used to attachat least one of the proteins to an N-linked glycan.

Both N-glycans and O-glycans are attached to proteins such as mAbs bythe cells producing these proteins. The cellular N-glycosylationmachinery recognises and glycosylates N-glycosylation signals (N—X—S/Tmotifs) in the amino acid chain, as the nascent protein is translocatedfrom the ribosome to the endoplasmic reticulum (Kiely et al., J. Biol.Chem. 1976, 251: 5490; Glabe et al., J. Biol. Chem., 1980, 255, 9236).Likewise, O-glycans are attached to specific O-glycosylation sites inthe amino acid chain, but the motifs triggering O-glycosylation are muchmore heterogeneous than the N-glycosylation signals, and our ability topredict O-glycosylation sites in amino acid sequences is stillinadequate (Julenius et al., Glycobiology, 2005, 15: 153). Methods ofconjugating polypeptides with various polymeric side groups aredescribed e.g. in WO0331464.

In another embodiment, a chemical moiety used in the linker comprises achemical moiety, which is used to attach said linker to at least one ofthe proteins with a structure selected of the biradicals:

Monospecific and bispecific antibodies of the current invention may behuman or humanised antibodies. The term “human antibody”, as usedherein, is intended to include antibodies having variable regions inwhich at least a portion of a framework region and/or at least a portionof a CDR region are derived from human germline immunoglobulinsequences. (For example, a human antibody may have variable regions inwhich both the framework and CDR regions are derived from human germlineimmunoglobulin sequences.) Furthermore, if the antibody contains aconstant region, the constant region or a portion thereof is alsoderived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo).

Such a human antibody may be a human monoclonal antibody. Such a humanmonoclonal antibody may be produced by a hybridoma which includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising human immunoglobulin heavy and lightchain gene segments repertoires, fused to an immortalised cell.

Human antibodies may be isolated from sequence libraries built onselections of human germline sequences, further diversified with naturaland synthetic sequence diversity.

Human antibodies may be prepared by in vitro immunisation of humanlymphocytes followed by transformation of the lymphocytes withEpstein-Barr virus.

The term “human antibody derivative” refers to any modified form of thehuman antibody, such as a conjugate of the antibody and another agent orantibody.

The term “humanised antibody”, as used herein, refers to ahuman/non-human chimeric antibody that contains a sequence (CDR regionsor parts thereof) derived from a non-human immunoglobulin. A humanisedantibody is, thus, a human immunoglobulin (recipient antibody) in whichat least residues from a hypervariable region of the recipient arereplaced by residues from a hypervariable region of an antibody from anon-human species (donor antibody) such as from a mouse, rat, rabbit ornon-human primate, which have the desired specificity, affinity,sequence composition and functionality. In some instances, framework(FR) residues of the human immunoglobulin are replaced by correspondingnon-human residues. An example of such a modification is theintroduction of one or more so-called back-mutations, which aretypically amino acid residues derived from the donor antibody.Humanisation of an antibody may be carried out using recombinanttechniques known to the person skilled in the art (see, e.g., AntibodyEngineering, Methods in Molecular Biology, vol. 248, edited by Benny K.Lo). A suitable human recipient framework for both the light and heavychain variable domain may be identified by, for example, sequence orstructural homology. Alternatively, fixed recipient frameworks may beused, e.g., based on knowledge of structure, biophysical and biochemicalproperties. The recipient frameworks can be germline derived or derivedfrom a mature antibody sequence. CDR regions from the donor antibody canbe transferred by CDR grafting. The CDR grafted humanised antibody canbe further optimised for e.g. affinity, functionality and biophysicalproperties by identification of critical framework positions wherere-introduction (backmutation) of the amino acid residue from the donorantibody has beneficial impact on the properties of the humanisedantibody. In addition to donor antibody derived backmutations, thehumanised antibody can be engineered by introduction of germlineresidues in the CDR or framework regions, elimination of immunogenicepitopes, site-directed mutagenesis, affinity maturation, etc.

Furthermore, humanised antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, a humanised antibody will comprise at least one—typicallytwo—variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and in whichall or substantially all of the FR residues are those of a humanimmunoglobulin sequence. The humanised antibody can, optionally, alsocomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin.

The term “humanised antibody derivative” refers to any modified form ofthe humanised antibody, such as a conjugate of the antibody and anotherchemical agent or antibody or antibody fragment or polypeptide.

The term “chimeric antibody”, as used herein, refers to an antibodywhose light and heavy chain genes have been constructed, typically bygenetic engineering, from immunoglobulin variable and constant regiongenes that originate from different species. For example, the variablesegments of genes from a mouse monoclonal antibody may be joined tohuman constant regions.

The fragment crystallisable region (“Fc region”/“Fc domain”) of anantibody is the C-terminal region of an antibody, which comprises theconstant CH2 and CH3 domains. The Fc domain may interact with cellsurface receptors called Fc receptors, as well as some proteins of thecomplement system. The Fc region enables antibodies to interact with theimmune system. In one aspect of the invention, antibodies may beengineered to include modifications within the Fc region, typically toalter one or more of its functional properties, such as serum half-life,complement fixation, Fc-receptor binding, protein stability and/orantigen-dependent cellular cytotoxicity, or lack thereof, among others.Furthermore, an antibody of the invention may be chemically modified(e.g., one or more chemical moieties can be attached to the antibody) orbe modified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. An IgG1 antibody may carry amodified Fc domain comprising one or more, and perhaps all of thefollowing mutations that will result in decreased affinity to certain Fcreceptors (L234A, L235E, and G237A) and in reduced C1q-mediatedcomplement fixation (A330S and P331S), respectively (residue numberingaccording to the EU index).

The isotype of an antibody of the invention may be IgG, such as IgG1,such as IgG2, such as IgG4. If desired, the class of an antibody may be“switched” by known techniques. For example, an antibody that wasoriginally produced as an IgM molecule may be class switched to an IgGantibody. Class switching techniques also may be used to convert one IgGsubclass to another, for example: from IgG1 to IgG2 or IgG4; from IgG2to IgG1 or IgG4; or from IgG4 to IgG1 or IgG2. Engineering of antibodiesto generate constant region chimeric molecules, by combination ofregions from different IgG subclasses, can also be performed.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further for instancein U.S. Pat. No. 5,677,425 by Bodmer et al.

The constant region may be modified to stabilise the antibody, e.g., toreduce the risk of a bivalent antibody separating into two monovalentVH-VL fragments. For example, in an IgG4 constant region, residue S228(according to the EU numbering index, S241 according to Kabat) may bemutated to a proline (P) residue to stabilise inter heavy chaindisulphide bridge formation at the hinge (see, e.g., Angal et al., MolImmunol. 1993; 30:105-8).

Bispecific antibodies or two monospecific antibodies that bind twounique epitopes on TFPI may be generated by methods known to a personskilled in the art. Bispecific formats may be prepared, for example, bychemical conjugation of two antibody fragments—such as two Fab or scFvfragments—either directly or via a linker providing the requiredflexibility for proper function. One specific method for coupling Fabfragments is to use the thiol functionality in cysteine residues placedappropriately in the Fab fragments.

Antibodies or fragments thereof may be defined in terms of theircomplementarity-determining regions (CDRs). The term“complementarity-determining region” or “hypervariable region”, whenused herein, refers to the regions of an antibody in which amino acidresidues involved in antigen-binding are situated. The region ofhypervariability or CDRs can be identified as the regions with thehighest variability in amino acid alignments of antibody variabledomains. Databases can be used for CDR identification such as the Kabatdatabase, the CDRs e.g. being defined as comprising amino acid residues24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light-chain variable domainand 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variabledomain; (Kabat et al., Sequences of Proteins of Immunological Interest,1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242) Alternatively CDRs can be defined as those residues from a“hypervariable loop” (residues 26-33 (L1), 50-52 (L2) and 91-96 (L3) inthe light-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101(H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol1987; 196: 901-917). Typically, the numbering of amino acid residues inthis region is performed by the method described in Kabat et al., supra.Phrases such as “Kabat position”, “Kabat residue”, and “according toKabat” herein refer to this numbering system for heavy chain variabledomains or light chain variable domains. Using the Kabat numberingsystem, the actual linear amino acid sequence of a peptide may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a framework (FR) or CDR of the variable domain. Forexample, a heavy chain variable domain may include amino acid insertions(residue 52a, 52b and 52c according to Kabat) after residue 52 of CDR H2and inserted residues (e.g. residues 82a, 82b, and 82c, etc. accordingto Kabat) after heavy chain FR residue 82. The Kabat numbering ofresidues may be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The term “framework region” or “FR” residues refer to those VH or VLamino acid residues that are not within the CDRs, as defined herein.

An antibody of the invention may comprise a CDR region from one or moreof the specific antibodies disclosed herein, such as a CDR region fromwithin SEQ ID NOs: 3 to 61, as defined using Kabat numbering oraccording to the sequential amino acid numbering disclosed herein.

-   -   Thus, a monospecific antibody of the invention which is capable        of binding TFPI (1-181) may have the portion of the bispecific        antibody of the invention which binds TFPI (1-181) may also        comprise said CDR regions.    -   A monospecific antibody of the invention which is capable of        binding TFPI (1-181) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID        NO: 3, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50 to 66 of SEQ ID        NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7, wherein one, two or three        of these amino acids may be substituted by a different amino        acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99 to 110 of SEQ ID        NO: 3, wherein one or two of these amino acid residues may be        substituted by a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (1-181) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24 to 39 of SEQ ID        NO: 4; and/or    -   a CDR2 sequence corresponding to amino acids 55 to 61 of SEQ ID        NO: 4; and/or    -   a CDR3 sequence corresponding to amino acids 94 to 102 of SEQ ID        NO: 4.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID        NO: 9, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50-66 of SEQ ID NO:        9, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99-107 of SEQ ID        NO: 9, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24-34 of SEQ ID NO:        10, wherein one or two of these amino acid residues may be        substituted with a different amino acid; and/or    -   a CDR2 sequence corresponding to amino acids 50-56 of SEQ ID NO:        10, wherein one of these amino acid residues may be substituted        with a different amino acid; and/or    -   a CDR3 sequence corresponding to amino acids 89-97 of SEQ ID NO:        10, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31-35 of SEQ ID NO:        11, wherein one of these amino acid residues may be substituted        by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50-66 of SEQ ID NO:        11, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99-106 of SEQ ID        NO: 11, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24-33 of SEQ ID NO:        12, wherein one or two of these amino acid residues may be        substituted with a different amino acid; and/or    -   a CDR2 sequence corresponding to amino acids 49-55 of SEQ ID NO:        12, wherein one of these amino acid residues may be substituted        with a different amino acid; and/or    -   a CDR3 sequence corresponding to amino acids 88-96 of SEQ ID NO:        12, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   The portion of the bispecific antibody of the invention which        binds TFPI (182-276) may also comprise said CDR regions.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31-35 of SEQ ID NO:        13, wherein one of these amino acid residues may be substituted        by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50-66 of SEQ ID NO:        13, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99-111 of SEQ ID        NO: 13, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24-38 of SEQ ID NO:        14, wherein one, two or three of these amino acid residues may        be substituted with a different amino acid; and/or    -   a CDR2 sequence corresponding to amino acids 54-60 of SEQ ID NO:        14, wherein one of these amino acid residues may be substituted        with a different amino acid; and/or    -   a CDR3 sequence corresponding to amino acids 93-101 of SEQ ID        NO: 14, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31-35 of SEQ ID NO:        15, wherein one of these amino acid residues may be substituted        by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50-66 of SEQ ID NO:        15, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99-106 of SEQ ID        NO: 15, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24-34 of SEQ ID NO:        16, wherein one or two of these amino acid residues may be        substituted with a different amino acid; and/or    -   a CDR2 sequence corresponding to amino acids 50-56 of SEQ ID NO:        16, wherein one of these amino acid residues may be substituted        with a different amino acid; and/or    -   a CDR3 sequence corresponding to amino acids 89-97 of SEQ ID NO:        16, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   The portion of the bispecific antibody of the invention which        binds TFPI (182-276) may comprise said CDR regions.    -   A monospecific antibody according to the invention which is        capable of binding TFPI (1-181) may have a heavy chain        comprising:    -   a CDR1 sequence corresponding to amino acids 31 to 36 of SEQ ID        NO: 22, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 51 to 66 of SEQ ID        NO: 22, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99 to 104 of SEQ ID        NO: 22, wherein one of these amino acid residues may be        substituted by a different amino acid.    -   A monospecific antibody according to the invention which is        capable of binding TFPI (1-181) may have a light chain        comprising:    -   a CDR1 sequence corresponding to amino acids 24 to 33 of SEQ ID        NO: 23, wherein one or two of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 49 to 55 of SEQ ID        NO: 23, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 88 to 96 of SEQ ID        NO: 23, wherein one of these amino acid residues may be        substituted by a different amino acid residue.    -   A monospecific antibody according to the invention which is        capable of binding TFPI (1-181) may have a heavy chain        comprising:    -   a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID        NO: 24, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID        NO: 24, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID        NO: 24, wherein one or two of these amino acid residues may be        substituted by a different amino acid.    -   A monospecific antibody according to the invention which is        capable of binding TFPI (1-181) may have a light chain        comprising:    -   a CDR1 sequence corresponding to amino acids 24 to 34 of SEQ ID        NO: 25, wherein one, two or three of these amino acid residues        may be substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50 to 56 of SEQ ID        NO: 25, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 89 to 96 of SEQ ID        NO: 25, wherein one of these amino acid residues may be        substituted by a different amino acid residue.    -   A monospecific antibody according to the invention which is        capable of binding TFPI (1-181) may have a heavy chain        comprising:    -   a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID        NO: 28, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50 to 66 of SEQ ID        NO: 28, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99 to 105 of SEQ ID        NO: 28, wherein one or two of these amino acid residues may be        substituted by a different amino acid.    -   A monospecific antibody according to the invention which is        capable of binding TFPI (1-181) may have a light chain        comprising:    -   a CDR1 sequence corresponding to amino acids 24 to 39 of SEQ ID        NO: 29, wherein one, two or three of these amino acid residues        may be substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 55 to 61 of SEQ ID        NO: 29, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 94 to 102 of SEQ ID        NO: 29, wherein one of these amino acid residues may be        substituted by a different amino acid residue.    -   An monospecific antibody according to the invention which is        capable of binding TFPI (1-181) may have a heavy chain        comprising:    -   a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID        NO: 30, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID        NO: 30, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID        NO: 30, wherein one or two of these amino acid residues may be        substituted by a different amino acid.    -   A monospecific antibody according to the invention which is        capable of binding TFPI (1-181) may have a light chain        comprising:    -   a CDR1 sequence corresponding to amino acids 24 to 34 of SEQ ID        NO: 31, wherein one, two or three of these amino acid residues        may be substituted by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50 to 56 of SEQ ID        NO: 31, wherein one of these amino acid residues may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 89 to 96 of SEQ ID        NO: 31, wherein one of these amino acid residues may be        substituted by a different amino acid residue.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31-35 of SEQ ID NO:        42, wherein one of these amino acid residues may be substituted        by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50-66 of SEQ ID NO:        42, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99-105 of SEQ ID        NO: 42, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24-38 of SEQ ID NO:        43, wherein one, two or three of these amino acid residues may        be substituted with a different amino acid; and/or    -   a CDR2 sequence corresponding to amino acids 54-60 of SEQ ID NO:        43, wherein one of these amino acid residues may be substituted        with a different amino acid; and/or    -   a CDR3 sequence corresponding to amino acids 93-101 of SEQ ID        NO: 43, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31-35 of SEQ ID NO:        44, wherein one of these amino acid residues may be substituted        by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50-66 of SEQ ID NO:        44, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99-109 of SEQ ID        NO: 44, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24-34 of SEQ ID NO:        45, wherein one, two or three of these amino acid residues may        be substituted with a different amino acid; and/or    -   a CDR2 sequence corresponding to amino acids 50-56 of SEQ ID NO:        45, wherein one of these amino acid residues may be substituted        with a different amino acid; and/or    -   a CDR3 sequence corresponding to amino acids 89-97 of SEQ ID NO:        45, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31-36 of SEQ ID NO:        46, wherein one of these amino acid residues may be substituted        by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 51-66 of SEQ ID NO:        46, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99-106 of SEQ ID        NO: 46, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24-38 of SEQ ID NO:        47, wherein one, two or three of these amino acid residues may        be substituted with a different amino acid; and/or    -   a CDR2 sequence corresponding to amino acids 54-60 of SEQ ID NO:        47, wherein one of these amino acid residues may be substituted        with a different amino acid; and/or    -   a CDR3 sequence corresponding to amino acids 93-101 of SEQ ID        NO: 47, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a heavy chain comprising:    -   a CDR1 sequence corresponding to amino acids 31-35 of SEQ ID NO:        60, wherein one of these amino acid residues may be substituted        by a different amino acid residue; and/or    -   a CDR2 sequence corresponding to amino acids 50-66 of SEQ ID NO:        60, wherein one, two or three of these amino acids may be        substituted by a different amino acid residue; and/or    -   a CDR3 sequence corresponding to amino acids 99-107 of SEQ ID        NO: 60, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   A monospecific antibody of the invention which is capable of        binding TFPI (182-276) may have a light chain comprising:    -   a CDR1 sequence corresponding to amino acids 24-34 of SEQ ID NO:        61, wherein one, two or three of these amino acid residues may        be substituted with a different amino acid; and/or    -   a CDR2 sequence corresponding to amino acids 50-56 of SEQ ID NO:        61, wherein one of these amino acid residues may be substituted        with a different amino acid; and/or    -   a CDR3 sequence corresponding to amino acids 89-97 of SEQ ID NO:        61, wherein one or two of these amino acid residues may be        substituted with a different amino acid.    -   Said monospecific antibodies may be combined to form a        KPI-1/KPI-3 bispecific antibody.

In one embodiment a KPI-1 domain specific antibody or fragment thereofis combined with C-terminal region (182-276 according to SEQ ID NO: 1)specific antibody or fragment thereof to form a bispecific antibody ininter alia full length format or Fab-Fab conjugate.

In one such embodiment mAb 1F91 (SEQ ID NOs: 22 and 23) or fragmentthereof is combined with mAb 22F74 (SEQ ID NO: 15 and 16) or fragmentthereof.

In one such embodiment mAb 1F91 (SEQ ID NOs: 22 and 23) or fragmentthereof is combined with mAb 22F132 (SEQ ID NO: 42 and 43) or fragmentthereof.

In one such embodiment mAb 1F91 (SEQ ID NOs: 22 and 23) or fragmentthereof is combined with mAb 22F79 (SEQ ID NO: 44 and 45) or fragmentthereof.

In one such embodiment mAb 1F91 (SEQ ID NOs: 22 and 23) or fragmentthereof is combined with mAb 41F41 (SEQ ID NO: 46 and 47) or fragmentthereof.

In one such embodiment mAb 1F91 (SEQ ID NOs: 22 and 23) or fragmentthereof is combined with mAb 41F30 (SEQ ID NO: 60 and 61) or fragmentthereof.

In one such embodiment mAb 2F3 (SEQ ID NOs: 24 and 25) or fragmentthereof is combined with mAb 22F74 (SEQ ID NO: 15 and 16) or fragmentthereof.

In one such embodiment mAb 2F3 (SEQ ID NOs: 24 and 25) or fragmentthereof is combined with mAb 22F132 (SEQ ID NO: 42 and 43) or fragmentthereof.

In one such embodiment mAb 2F3 (SEQ ID NOs: 24 and 25) or fragmentthereof is combined with mAb 22F79 (SEQ ID NO: 44 and 45) or fragmentthereof.

In one such embodiment mAb 2F3 (SEQ ID NOs: 24 and 25) or fragmentthereof is combined with mAb 41F41 (SEQ ID NO: 46 and 47) or fragmentthereof.

In one such embodiment mAb 2F3 (SEQ ID NOs: 24 and 25) or fragmentthereof is combined with mAb 41F30 (SEQ ID NO: 60 and 61) or fragmentthereof.

In one such embodiment mAb 2F22 (SEQ ID NOs: 26 and 27) or fragmentthereof is combined with mAb 22F74 (SEQ ID NO: 15 and 16) or fragmentthereof.

In one such embodiment mAb 2F22 (SEQ ID NOs: 26 and 27) or fragmentthereof is combined with mAb 22F132 (SEQ ID NO: 42 and 43) or fragmentthereof.

In one such embodiment mAb 2F22 (SEQ ID NOs: 26 and 27) or fragmentthereof is combined with mAb 22F79 (SEQ ID NO: 44 and 45) or fragmentthereof.

In one such embodiment mAb 2F22 (SEQ ID NOs: 26 and 27) or fragmentthereof is combined with mAb 41F41 (SEQ ID NO: 46 and 47) or fragmentthereof.

In one such embodiment mAb 2F22 (SEQ ID NOs: 26 and 27) or fragmentthereof is combined with mAb 41F30 (SEQ ID NO: 60 and 61) or fragmentthereof.

In one such embodiment mAb 2F35 (SEQ ID NOs: 28 and 29) or fragmentthereof is combined with mAb 22F74 (SEQ ID NO: 15 and 16) or fragmentthereof.

In one such embodiment mAb 2F35 (SEQ ID NOs: 28 and 29) or fragmentthereof is combined with mAb 22F132 (SEQ ID NO: 42 and 43) or fragmentthereof.

In one such embodiment mAb 2F35 (SEQ ID NOs: 28 and 29) or fragmentthereof is combined with mAb 22F79 (SEQ ID NO: 44 and 45) or fragmentthereof.

In one such embodiment mAb 2F35 (SEQ ID NOs: 28 and 29) or fragmentthereof is combined with mAb 41F41 (SEQ ID NO: 46 and 47) or fragmentthereof.

In one such embodiment mAb 2F35 (SEQ ID NOs: 28 and 29) or fragmentthereof is combined with mAb 41F30 (SEQ ID NO: 60 and 61) or fragmentthereof.

In one such embodiment mAb 2F45 (SEQ ID NOs: 30 and 31) or fragmentthereof is combined with mAb 22F74 (SEQ ID NO: 15 and 16) or fragmentthereof.

In one such embodiment mAb 2F45 (SEQ ID NOs: 30 and 31) or fragmentthereof is combined with mAb 22F132 (SEQ ID NO: 42 and 43) or fragmentthereof.

In one such embodiment mAb 2F45 (SEQ ID NOs: 30 and 31) or fragmentthereof is combined with mAb 22F79 (SEQ ID NO: 44 and 45) or fragmentthereof.

In one such embodiment mAb 2F45 (SEQ ID NOs: 30 and 31) or fragmentthereof is combined with mAb 41F41 (SEQ ID NO: 46 and 47) or fragmentthereof.

In one such embodiment mAb 2F45 (SEQ ID NOs: 30 and 31) or fragmentthereof is combined with mAb 41F30 (SEQ ID NO: 60 and 61) or fragmentthereof.

A variant antibody may comprise 1, 2, 3, 4, 5, up to 10 or more aminoacid substitutions and/or deletions and/or insertions from the specificsequences and fragments discussed above. “Deletion” variants maycomprise the deletion of individual amino acids, deletion of smallgroups of amino acids such as 1, 2, 3, 4 or 5 amino acids, or deletionof larger amino acid regions, such as the deletion of specific aminoacid domains or other features. “Insertion” variants may comprise theinsertion of individual amino acids, insertion of small groups of aminoacids such as 1, 2, 3, 4 or 5 amino acids, or insertion of larger aminoacid regions, such as the insertion of specific amino acid domains orother features. “Substitution” variants preferably involve thereplacement of one or more amino acids with the same number of aminoacids and making conservative amino acid substitutions. For example, anamino acid may be substituted with an alternative amino acid havingsimilar properties, for example, another basic amino acid, anotheracidic amino acid, another neutral amino acid, another charged aminoacid, another hydrophilic amino acid, another hydrophobic amino acid,another polar amino acid, another aromatic amino acid or anotheraliphatic amino acid. Some properties of the 20 main amino acids whichcan be used to select suitable substituents are as follows:

Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral Cys polar,hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar,hydrophilic, charged (−) Pro hydrophobic, neutral Glu polar,hydrophilic, charged (−) Gln polar, hydrophilic, neutral Phe aromatic,hydrophobic, neutral Arg polar, hydrophilic, charged Gly aliphatic,neutral (+) His aromatic, polar, hydrophilic, Ser polar, hydrophilic,neutral charged (+) Thr polar, hydrophilic, neutral Ile aliphatic,hydrophobic, neutral Val aliphatic, hydrophobic, Lys polar, hydrophilic,charged (+) neutral Leu aliphatic, hydrophobic, neutral Trp aromatic,hydrophobic, neutral Tyr aromatic, polar, hydrophobic

Preferred “derivatives” or “variants” include those in which instead ofthe naturally occurring amino acid the amino acid which appears in thesequence is a structural analogue thereof. Amino acids used in thesequences may also be derivatized or modified, e.g. labelled, providingthe function of the antibody is not significantly adversely affected.

Substitutions may be, but are not limited to, conservativesubstitutions.

Derivatives and variants as described above may be prepared duringsynthesis of the antibody or by post-production modification, or whenthe antibody is in recombinant form using the known techniques ofsite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids.

The invention also relates to polynucleotides that encode antibodies ofthe invention. Thus, a polynucleotide of the invention may encode anyantibody as described herein. The terms “nucleic acid molecule” and“polynucleotide” are used interchangeably herein and refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogues thereof. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, messenger RNA (mRNA),cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA ofany sequence, isolated RNA of any sequence, nucleic acid probes, andprimers. A polynucleotide of the invention may be provided in isolatedor purified form.

A nucleic acid sequence which “encodes” a selected polypeptide is anucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

In one embodiment, a polynucleotide of the invention comprises asequence which encodes a VH or VL amino acid sequence as describedabove. For example, a polynucleotide of the invention may encode apolypeptide comprising the sequence of SEQ ID NO: 11, or a variant orfragment thereof as described above. A suitable polynucleotide sequencemay alternatively be a variant of one of these specific polynucleotidesequences. For example, a variant may be a substitution, deletion oraddition variant of any of the above nucleic acid sequences. A variantpolynucleotide may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30,up to 40, up to 50, up to 75 or more nucleic acid substitutions and/ordeletions from the sequences given in the sequence listing.

Suitable variants may be at least 70% homologous to a polynucleotideencoding a polypeptide of the present invention preferably at least 80or 90% and more preferably at least 95%, 97% or 99% homologous thereto.Methods of measuring homology are well known in the art and it will beunderstood by those of skill in the art that in the present context,homology is calculated on the basis of nucleic acid identity. Suchhomology may exist over a region of at least 15, preferably at least 30,for instance at least 40, 60, 100, 200 or more contiguous nucleotides.Such homology may exist over the entire length of the unmodifiedpolynucleotide sequence.

Methods of measuring polynucleotide homology or identity are known inthe art. For example, the UWGCG Package provides the BESTFIT programwhich can be used to calculate homology (e.g. used on its defaultsettings) (Devereux et al. (1984) Nucleic Acids Research 12: 387-395).

The PILEUP and BLAST algorithms can also be used to calculate homologyor line up sequences (typically on their default settings), for exampleas described in Altschul S. F. (1993) J Mol Evol 36: 290-300; Altschul,S. F. et al. (1990) J Mol Biol 215: 403-10.

Software for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al. supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, anda comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787.

The term “antigen” (Ag) herein refers to the entity used forimmunisation of an immunocompetent vertebrate to produce an antibody(Ab) that recognises the Ag. In the context of the current invention,suitable antigens include human TFPI (1-181) and full length humanTFPIα. Herein, Ag is termed more broadly and is generally intended toinclude target molecules that are specifically recognised by the Ab,thus including fragments or mimics of the molecule used in theimmunisation process, or other process, e.g. phage display, used forgenerating the Ab.

The term “epitope”, as used herein, is defined in the context of amolecular interaction between an “antigen-binding polypeptide”, such asan antibody (Ab), and its corresponding antigen (Ag). Generally,“epitope” refers to the area or region on an Ag to which an Abspecifically binds, i.e. the area or region in physical contact with theAb. Physical contact may be defined using various criteria (e.g. adistance cut-off of 2-6 Å, such as 3 Å, such as 4 Å, such as 5 Å; orsolvent accessibility) for atoms in the Ab and Ag molecules. A proteinepitope may comprise amino acid residues in the Ag that are directlyinvolved in binding to a Ab (also called the immunodominant component ofthe epitope) and other amino acid residues, which are not directlyinvolved in binding, such as amino acid residues of the Ag which areeffectively blocked by the Ab, i.e. amino acid residues within the“solvent-excluded surface” and/or the “footprint” of the Ab.

The term “epitope” herein comprises both types of binding region in anyparticular region of TFPI that specifically binds to a mono- orbispecific TFPI antibody, or another TFPI-specific agent according tothe invention, unless otherwise stated. TFPI may comprise a number ofdifferent epitopes, which may include, without limitation, (1) linearpeptide epitopes (2) conformational epitopes which consist of one ormore non-contiguous amino acids located near each other in the matureTFPI conformation; and (3) post-translational epitopes which consist,either in whole or part, of molecular structures covalently attached toTFPI, such as carbohydrate groups.

The epitope for a given antibody (Ab)/antigen (Ag) pair can be describedand characterised at different levels of detail using a variety ofexperimental and computational epitope mapping methods. The experimentalmethods include mutagenesis, X-ray crystallography, Nuclear MagneticResonance (NMR) spectroscopy, Hydrogen deuterium eXchange MassSpectrometry (HX-MS) and various competition binding methods; methodsthat are known in the art. As each method relies on a unique principle,the description of an epitope is intimately linked to the method bywhich it has been determined. Thus, depending on the epitope mappingmethod employed, the epitope for a given Ab/Ag pair may be describeddifferently.

At its most detailed level, the epitope for the interaction between theAg and the Ab can be described by the spatial coordinates defining theatomic contacts present in the Ag-Ab interaction, as well as informationabout their relative contributions to the binding thermodynamics. At aless detailed level, the epitope can be characterised by the spatialcoordinates defining the atomic contacts between the Ag and Ab. At aneven less detailed level the epitope can be characterised by the aminoacid residues that it comprises as defined by a specific criteria suchas the distance between or solvent accessibility of atoms in the Ab:Agcomplex. At a further less detailed level the epitope can becharacterised through function, e.g. by competition binding with otherAbs. The epitope can also be defined more generically as comprisingamino acid residues for which substitution by another amino acid willalter the characteristics of the interaction between the Ab and Ag.

In the context of an X-ray derived crystal structure defined by spatialcoordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag,the term epitope is herein, unless otherwise specified or contradictedby context, specifically defined as being TFPI residues having a heavyatom (i.e. a non-hydrogen atom) within a distance of 4 Å from a heavyatom in the Ab.

From the fact that descriptions and definitions of epitopes, dependanton the epitope mapping method used, are obtained at different levels ofdetail, it follows that comparison of epitopes for different Abs on thesame Ag can similarly be conducted at different levels of detail.

Epitopes described at the amino acid level, e.g. determined from anX-ray structure, are said to be identical if they contain the same setof amino acid residues. Epitopes are said to overlap if at least oneamino acid is shared by the epitopes. Epitopes are said to be separate(unique) if no amino acid residue is shared by the epitopes.

The definition of the term “paratope” is derived from the abovedefinition of “epitope” by reversing the perspective. Thus, the term“paratope” refers to the area or region on the Ab to which an Agspecifically binds, i.e. with which it makes physical contact to the Ag.

In the context of an X-ray derived crystal structure, defined by spatialcoordinates of a complex between an Ab, such as a Fab fragment, and itsAg, the term paratope is herein, unless otherwise specified orcontradicted by context, specifically defined as Ag residuescharacterised by having a heavy atom (i.e. a non-hydrogen atom) within adistance of 4 Å from a heavy atom in TFPI.

The epitope and paratope for a given antibody (Ab)/antigen (Ag) pair maybe identified by routine methods. For example, the general location ofan epitope may be determined by assessing the ability of an antibody tobind to different fragments or variant TFPI polypeptides. The specificamino acids within TFPI that make contact with an antibody (epitope) andthe specific amino acids in an antibody that make contact with TFPI(paratope) may also be determined using routine methods. For example,the antibody and target molecule may be combined and the Ab:Ag complexmay be crystallised. The crystal structure of the complex may bedetermined and used to identify specific sites of interaction betweenthe antibody and its target.

Antibodies that bind to the same antigen can be characterised withrespect to their ability to bind to their common antigen simultaneouslyand may be subjected to “competition binding”/“binning”. In the presentcontext, the term “binning” refers to a method of grouping antibodiesthat bind to the same antigen. “Binning” of antibodies may be based oncompetition binding of two antibodies to their common antigen in assaysbased on standard techniques such as surface plasmon resonance (SPR),ELISA or flow cytometry.

An antibody's “bin” is defined using a reference antibody. If a secondantibody is unable to bind to an antigen at the same time as thereference antibody, the second antibody is said to belong to the same“bin” as the reference antibody. In this case, the reference and thesecond antibody competitively bind the same part of an antigen and arecoined “competing antibodies”. If a second antibody is capable ofbinding to an antigen at the same time as the reference antibody, thesecond antibody is said to belong to a separate “bin”. In this case, thereference and the second antibody do not competitively bind the samepart of an antigen and are coined “non-competing antibodies”.

Antibody “binning” does not provide direct information about theepitope. Competing antibodies, i.e. antibodies belonging to the same“bin” may have identical epitopes, overlapping epitopes or even separateepitopes. The latter is the case if the reference antibody bound to itsepitope on the antigen takes up the space required for the secondantibody to contact its epitope on the antigen (“steric hindrance”).Non-competing antibodies generally have separate epitopes.

Adjusting the relative and absolute affinities of the two antigenrecognition sites of the monospecific antibodies or the bispecificantibody for TFPI (1-181) and TFPI (182-276) may be advantageous interms of, for example, providing selective binding to TFPIα while stillproviding complete blockage of TFPI inhibition.

The term “binding affinity” is herein used as a measure of the strengthof a non-covalent interaction between two molecules, e.g. an antibody,or fragment thereof, and an antigen. The term “binding affinity” is usedto describe monovalent interactions (intrinsic activity).

Binding affinity between two molecules, e.g. an antibody, or fragmentthereof, and an antigen, through a monovalent interaction may bequantified by determining the equilibrium dissociation constant (K_(D)).In turn, K_(D) can be determined by measurement of the kinetics ofcomplex formation and dissociation, e.g. by the SPR method. The rateconstants corresponding to the association and the dissociation of amonovalent complex are referred to as the association rate constantk_(a) (or k_(on)) and dissociation rate constant k_(d) (or k_(off)),respectively. K_(D) is related to k_(a) and k_(d) through the equationK_(D)=k_(d)/k_(a).

Following the above definition, binding affinities associated withdifferent molecular interactions, such as comparison of the bindingaffinity of different antibodies for a given antigen, may be compared bycomparison of the K_(D) values for the individual antibody/antigencomplexes.

An antibody according to the current invention may be able to competewith another molecule, such as a naturally occurring ligand or receptoror another antibody, for binding to TFPI and thereby affect functionsassociated with these interactions. The ability of an antibody tocompete with a natural ligand/receptor may be assessed by variousactivity assays measuring the effect on the apparent K_(I) for TFPIinhibition. K_(D) values may then be deduced from apparent K_(I) values.Typically, the K_(D) value of interest for the antibody with respect tothe target (TFPI) will be 2-fold, preferably 5-fold, more preferably10-fold lower than the K_(D) of other TFPI ligands. More preferably, theK_(D) will be 50-fold less, such as 100-fold less, or 200-fold less;even more preferably 500-fold less, such as 1,000-fold less, or10,000-fold less.

The value of this dissociation constant can be determined directly bywell-known methods. Standard assays to evaluate the binding ability ofligands such as antibodies towards targets are known in the art andinclude, for example, ELISAs, Western blots, RIAs, isothermal titrationcalorimetry, and flow cytometry analysis. The binding kinetics andbinding affinity of the antibody also can be assessed by standard assaysknown in the art, such as SPR.

A competitive binding assay can be conducted in which the binding of theantibody to the target is compared to the binding of the target byanother ligand of that target, such as another antibody.

A monospecific antibody, or any arm of a bispecific antibody of theinvention, may have a K_(D) for its target of 1×10⁻⁷ M or less, 1×10⁻⁸ Mor less, or 1×10⁻⁹ M or less, or 1×10⁻¹⁰ M or less, 1×10⁻¹¹ M or less,or 1×10⁻¹² M or less. The K_(D) of an antibody of the current inventionmay be less than 0.8 nM, such as less than 0.7 nM, such as less than 0.6nM, such as less than 0.5 nM, such as less than 0.4 nM, such as lessthan 0.3 nM, such as less than 0.2 nM, such as less than 0.1 nM, such asless than 0.05 nM, such as less than 0.025 nM, such as less than 0.015nM, such as between 0.015 nM and 0 nM.

In one aspect the present invention comprises antibodies that competewith the antibodies of the present invention to the extent that suchcompeting antibodies are bispecific.

In another aspect, the present invention provides compositions andformulations comprising molecules of the invention, such as thebispecific antibodies described herein. For example, the inventionprovides a pharmaceutical composition that comprises one or morebispecific TFPI antibodies of the invention, formulated together with apharmaceutically acceptable carrier.

Accordingly, one object of the invention is to provide a pharmaceuticalformulation comprising such a bispecific TFPI antibody which is presentin a concentration from 0.25 mg/ml to 250 mg/ml, and wherein saidformulation has a pH from 2.0 to 10.0. The formulation may furthercomprise one or more of a buffer system, a preservative, a tonicityagent, a chelating agent, a stabiliser, or a surfactant, as well asvarious combinations thereof. The use of preservatives, isotonic agents,chelating agents, stabilisers and surfactants in pharmaceuticalcompositions is well-known to the skilled person. Reference may be madeto Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In one embodiment, the pharmaceutical formulation is an aqueousformulation. Such a formulation is typically a solution or a suspension,but may also include colloids, dispersions, emulsions, and multi-phasematerials. The term “aqueous formulation” is defined as a formulationcomprising at least 50% w/w water. Likewise, the term “aqueous solution”is defined as a solution comprising at least 50% w/w water, and the term“aqueous suspension” is defined as a suspension comprising at least 50%w/w water.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, to which the physician or the patient adds solvents and/ordiluents prior to use.

In a further aspect, the pharmaceutical formulation comprises an aqueoussolution of such an antibody, and a buffer, wherein the antibody ispresent in a concentration from 1 mg/ml or above, and wherein saidformulation has a pH from about 2.0 to about 10.0.

A bispecific antibody or a pharmaceutical formulation comprising twomonospecific antibodies according to the invention may be used to treata subject with a coagulopathy.

The term “subject”, as used herein, includes any human patient, ornon-human vertebrate.

The term “coagulopathy”, as used herein, refers to an increasedhaemorrhagic tendency which may be caused by any qualitative orquantitative deficiency of any pro-coagulative component of the normalcoagulation cascade, or any upregulation of fibrinolysis. Suchcoagulopathies may be congenital and/or acquired and/or iatrogenic andare identified by a person skilled in the art.

Non-limiting examples of congenital hypocoagulopathies are haemophiliaA, haemophilia B, Factor VII deficiency, Factor X deficiency, Factor XIdeficiency, von Willebrand's disease and thrombocytopenias such asGlanzmann's thombasthenia and Bernard-Soulier syndrome. Said haemophiliaA or B may be severe, moderate or mild. The clinical severity ofhaemophilia is determined by the concentration of functional units ofFIX/FVIII in the blood and is classified as mild, moderate, or severe.Severe haemophilia is defined by a clotting factor level of <0.01 U/mlcorresponding to <1% of the normal level, while moderate and mildpatients have levels from 1-5% and >5%, respectively. Haemophilia A with“inhibitors” (that is, allo-antibodies against factor VIII) andhaemophilia B with “inhibitors” (that is, allo-antibodies against factorIX) are non-limiting examples of coagulopathies that are partlycongenital and partly acquired.

A non-limiting example of an acquired coagulopathy is serine proteasedeficiency caused by vitamin K deficiency; such vitamin K-deficiency maybe caused by administration of a vitamin K antagonist, such as warfarin.Acquired coagulopathy may also occur following extensive trauma. In thiscase otherwise known as the “bloody vicious cycle”, it is characterisedby haemodilution (dilutional thrombocytopaenia and dilution of clottingfactors), hypothermia, consumption of clotting factors and metabolicderangements (acidosis). Fluid therapy and increased fibrinolysis mayexacerbate this situation. Said haemorrhage may be from any part of thebody.

A non-limiting example of an iatrogenic coagulopathy is an overdosage ofanticoagulant medication—such as heparin, aspirin, warfarin and otherplatelet aggregation inhibitors—that may be prescribed to treatthromboembolic disease. A second, non-limiting example of iatrogeniccoagulopathy is that which is induced by excessive and/or inappropriatefluid therapy, such as that which may be induced by a blood transfusion.

In one embodiment of the current invention, haemorrhage is associatedwith haemophilia A or B. In another embodiment, haemorrhage isassociated with haemophilia A or B with acquired inhibitors. In anotherembodiment, haemorrhage is associated with thrombocytopenia. In anotherembodiment, haemorrhage is associated with von Willebrand's disease. Inanother embodiment, haemorrhage is associated with severe tissue damage.In another embodiment, haemorrhage is associated with severe trauma. Inanother embodiment, haemorrhage is associated with surgery. In anotherembodiment, haemorrhage is associated with haemorrhagic gastritis and/orenteritis. In another embodiment, the haemorrhage is profuse uterinebleeding, such as in placental abruption. In another embodiment,haemorrhage occurs in organs with a limited possibility for mechanicalhaemostasis, such as intracranially, intraaurally or intraocularly. Inanother embodiment, haemorrhage is associated with anticoagulanttherapy.

The term “treatment”, as used herein, refers to the medical therapy ofany human or other vertebrate subject in need thereof. Said subject isexpected to have undergone physical examination by a medicalpractitioner, or a veterinary medical practitioner, who has given atentative or definitive diagnosis which would indicate that the use ofsaid specific treatment is beneficial to the health of said human orother vertebrate. The timing and purpose of said treatment may vary fromone individual to another, according to the status quo of the subject'shealth. Thus, said treatment may be prophylactic, palliative,symptomatic and/or curative. In terms of the present invention,prophylactic, palliative, symptomatic and/or curative treatments mayrepresent separate aspects of the invention.

An antibody of the invention may be administered parenterally, such asintravenously, such as intramuscularly, such as subcutaneously.Alternatively, an antibody of the invention may be administered via anon-parenteral route, such as perorally or topically. An antibody of theinvention may be administered prophylactically. An antibody of theinvention may be administered therapeutically (on demand).

In one embodiment the bispecific antibodies of the present invention areused to measure TFPIα levels in vitro.

The following are non-limiting aspects of the invention:

-   -   1. A multispecific antibody that is capable of specifically        binding tissue factor pathway inhibitor (TFPI).    -   2. A multispecific antibody that is capable of specifically        binding a first epitope of tissue factor pathway inhibitor        (TFPI) and a second epitope of TFPI, wherein the first epitope        comprises an amino acid residue located within positions 1 to        181 of SEQ ID NO: 1 and the second epitope comprises an amino        acid residue located within TFPI positions 182 to 276 of SEQ ID        NO: 1.    -   3. The multispecific antibody according to aspects 1 or 2,        wherein the first epitope comprises an amino acid residue within        the KPI-1 domain of tissue factor pathway inhibitor (TFPI).    -   4. The multispecific antibody according to aspects 1 or 2,        wherein the first epitope comprises an amino acid residue within        the KPI-2 domain of tissue factor pathway inhibitor (TFPI).    -   5. The multispecific antibody according to any one of aspects 1        to 4, wherein the second epitope comprises an amino acid residue        within the KPI-3 domain of tissue factor pathway inhibitor        (TFPI).    -   6. A multispecific antibody according to any one of aspects 1 to        5 which is a bispecific antibody.    -   7. The bispecific antibody according to aspect 6 that is capable        of specifically binding tissue factor pathway inhibitor (TFPI),        wherein said antibody is capable of binding a KPI-1 epitope        comprising one or more amino acid residues selected from the        group consisting of Leu 16, Pro 17, Leu 19, Lys 20, Leu 21, Met        22, Phe 25, Cys 35, Ala 37, Met 39, Arg 41, Tyr 56, Gly 57, Gly        58, Cys 59, Glu 60, Gly 61, Asn 62, Gln 63, Arg 65, Phe 66, Glu        67, Glu 71 and Met 75 of SEQ ID NO: 1    -   8. The bispecific antibody according to aspect 7, wherein said        antibody is capable of binding a KPI-1 epitope comprising amino        acid residues Arg 41, Arg 65 and/or Glu 67 of SEQ ID NO: 1.    -   9. A bispecific antibody according to aspect 6 that is capable        of specifically binding tissue factor pathway inhibitor (TFPI),        wherein said antibody is capable of binding a KPI-2 epitope        comprising one or more amino acid residues selected from the        group consisting of Glu 100, Glu 101, Asp 102, Pro 103, Arg 107,        Tyr 109, Thr 111, Tyr 113, Phe 114, Asn 116, Gln 118, Gln 121,        Cys 122, Glu 123, Arg 124, Phe 125, Lys 126 and Leu 140 of SEQ        ID NO: 1.    -   10. The bispecific antibody according to aspect 9 that is        capable of specifically binding tissue factor pathway inhibitor        (TFPI) wherein said antibody is capable of specifically binding        a KPI-2 epitope comprising amino acid residue Arg 107 of SEQ ID        NO: 1.    -   11. The bispecific antibody according to any one of aspects 6 to        10 that is capable of specifically binding tissue factor pathway        inhibitor (TFPI), wherein said antibody is capable of binding a        KPI-3 epitope comprising one or more amino acid residues        selected from the group consisting of Tyr 207, Asn 208, Ser 209,        Val 210, Ile 211, Gly 212, Lys 213, Arg 215, Lys 232, Gln 233,        Leu 236 and Lys 240 of SEQ ID NO: 1.    -   12. The bispecific antibody according to aspect 11 wherein said        antibody is capable of binding a KPI-3 epitope comprising amino        acid residue Ile 211, Lys 213 and/or Arg 215 of SEQ ID NO: 1.    -   13. The bispecific antibody according to any one of aspects 6 to        8 that is capable of specifically binding tissue factor pathway        inhibitor (TFPI), wherein said antibody is capable of binding a        KPI-1 epitope comprising one or more amino acid residues        selected from the group consisting of Leu 16, Pro 17, Leu 19,        Lys 20, Leu 21, Met 22, Phe 25, Cys 35, Ala 37, Met 39, Arg 41,        Tyr 56, Gly 57, Gly 58, Cys 59, Glu 60, Gly 61, Asn 62, Gln 63,        Arg 65, Phe 66, Glu 67, Glu 71 and Met 75 of SEQ ID NO: 1; and    -   a KPI-3 epitope comprising one or more amino acid residues        selected from the group consisting of Tyr 207, Asn 208, Ser 209,        Val 210, Ile 211, Gly 212, Lys 213, Arg 215, Lys 232, Gln 233,        Leu 236 and Lys 240 of SEQ ID NO: 1.    -   14. The bispecific antibody according to any one of aspects 9 to        12 that is capable of specifically binding tissue factor pathway        inhibitor (TFPI), wherein said antibody is capable of binding a        KPI-2 epitope comprising one or more amino acid residues        selected from the group consisting of Glu 100, Glu 101, Asp 102,        Pro 103, Arg 107, Tyr 109, Thr 111, Tyr 113, Phe 114, Asn 116,        Gln 118, Gln 121, Cys 122, Glu 123, Arg 124, Phe 125, Lys 126        and Leu 140 of SEQ ID NO: 1; and a KPI-3 epitope comprising one        or more amino acid residues selected from the group consisting        of Tyr 207, Asn 208, Ser 209, Val 210, Ile 211, Gly 212, Lys        213, Arg 215, Lys 232, Gln 233, Leu 236 and Lys 240 of SEQ ID        NO: 1.    -   15. The bispecific antibody according to aspect 6 comprising a        heavy chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 99 to            110 of SEQ ID NO: 3;        -   and a light chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 94 to            102 of SEQ ID NO: 4;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted by a different        amino acid.    -   16. The bispecific antibody according to aspect 15, wherein the        heavy chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 3; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of a sequence selected from the group consisting of SEQ            ID NO: 3 or SEQ ID NO: 5 and SEQ ID NO: 7;        -   and the light chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 24 to            39 of SEQ ID NO: 4; and        -   a CDR2 sequence corresponding to amino acid residues 55 to            61 of SEQ ID NO: 4;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted by a different        amino acid.    -   17. The antibody according to aspect 6, comprising a heavy chain        comprising:        -   a CDR3 sequence corresponding to amino acid residues 99 to            107 of SEQ ID NO: 9;        -   and a light chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 89 to            97 of SEQ ID NO: 10;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   18. The bispecific antibody according to aspect 17, wherein the        heavy chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 9; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of SEQ ID NO: 9;    -   and wherein the light chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 24 to            34 of SEQ ID NO: 10;        -   a CDR2 sequence corresponding to amino acid residues 50 to            56 of SEQ ID NO: 10;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   19. The bispecific antibody according to aspect 6, comprising a        heavy chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 99 to            106 of SEQ ID NO: 11;    -   and a light chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 88 to            96 of SEQ ID NO: 12;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   20. The bispecific antibody according to aspect 19, wherein the        heavy chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 11; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of SEQ ID NO: 11;    -   and wherein the light chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 24 to            33 of SEQ ID NO: 12; and        -   a CDR2 sequence corresponding to amino acid residues 49 to            55 of SEQ ID NO: 12;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   21. The bispecific antibody according to aspect 6, comprising a        heavy chain comprising        -   a CDR3 sequence corresponding to amino acid residues 99 to            111 of SEQ ID NO: 13;    -   and a light chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 93 to            101 of SEQ ID NO: 14;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   22. The bispecific antibody according to aspect 21, wherein the        heavy chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 13; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of SEQ ID NO: 13;    -   and wherein the light chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 24 to            38 of SEQ ID NO: 14; and        -   a CDR2 sequence corresponding to amino acid residues 54 to            60 of SEQ ID NO: 14;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   23. The bispecific antibody according to aspect 6, comprising a        heavy chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 99 to            106 of SEQ ID NO: 15;    -   and a light chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 89 to            97 of SEQ ID NO: 16;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   24. The bispecific antibody according to aspect 23, wherein the        heavy chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 15; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of SEQ ID NO: 15;    -   and wherein the light chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 24 to            34 of SEQ ID NO: 16; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            56 of SEQ ID NO: 16;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   25. The bispecific antibody according to aspect 6, comprising a        heavy chain comprising        -   a CDR3 sequence corresponding to amino acid residues 98 to            110 of SEQ ID NO: 26;    -   and a light chain comprising:        -   a CDR3 sequence corresponding to amino acid residues 89 to            96 of SEQ ID NO: 27;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   26. The bispecific antibody according to aspect 25, wherein the        heavy chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 26; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            65 of SEQ ID NO: 26;    -   and wherein the light chain further comprises:        -   a CDR1 sequence corresponding to amino acid residues 24 to            34 of SEQ ID NO: 27; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            56 of SEQ ID NO: 27;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   27. A bispecific antibody according to aspect 6 which is in a        full length antibody format.    -   28. A bispecific antibody according to any one of aspects 6 to        31, which is a Fab-Fab conjugate or fusion protein.    -   29. The Fab-Fab conjugate according to aspect 28 comprising a        first Fab and a second Fab, wherein the first Fab comprises a        first heavy chain (HC) and a first light chain (LC), the first        heavy chain comprising:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 3;        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of a sequence selected from a group consisting of SEQ ID            NO: 3 or SEQ ID NO: 5 and SEQ ID NO: 7; and        -   a CDR3 sequence corresponding to amino acid residues 99 to            110 of SEQ ID NO: 3;    -   and the first light chain (LC) comprising        -   a CDR1 sequence corresponding to amino acid residues 24 to            39 of SEQ ID NO: 4;        -   a CDR2 sequence corresponding to amino acid residues 55 to            61 of SEQ ID NO: 4; and        -   a CDR3 sequence corresponding to amino acid residues 94 to            102 of SEQ ID NO: 4;    -   and the second Fab comprising a second heavy chain and a second        light chain, the second heavy chain (HC) comprising:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 9;        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of SEQ ID NO: 9; and        -   a CDR3 sequence corresponding to amino acid residues 99 to            107 of SEQ ID NO: 9;    -   and the second light chain (LC) comprising:        -   a CDR1 sequence corresponding to amino acid residues 24 to            34 of SEQ ID NO: 10;        -   a CDR2 sequence corresponding to amino acid residues 50 to            56 of SEQ ID NO: 10; and        -   a CDR3 sequence corresponding to amino acid residues 89 to            97 of SEQ ID NO: 10;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted by a different        amino acid.    -   30. The Fab-Fab conjugate according to aspect 29 comprising a        first Fab and a second Fab, wherein the first Fab fragment        comprises a first heavy chain (HC) and a first light chain (LC),        the first heavy chain comprising:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 26;        -   a CDR2 sequence corresponding to amino acid residues 50 to            65 of SEQ ID NO: 26; and        -   a CDR3 sequence corresponding to amino acid residues 98 to            110 of SEQ ID NO: 26;    -   and the first light chain (LC) comprising:        -   a CDR1 sequence corresponding to amino acid residues 24 to            34 of SEQ ID NO: 27;        -   a CDR2 sequence corresponding to amino acid residues 50 to            56 of SEQ ID NO: 27; and        -   a CDR3 sequence corresponding to amino acid residues 89 to            96 of SEQ ID NO: 27;    -   and the second Fab fragment comprising a second heavy chain and        a second light chain, the second heavy chain (HC) comprising:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 9;        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of SEQ ID NO: 9; and        -   a CDR3 sequence corresponding to amino acid residues 99 to            107 of SEQ ID NO: 9;    -   and the second light chain (LC) comprising:        -   a CDR1 sequence corresponding to amino acid residues 24 to            34 of SEQ ID NO: 10;        -   a CDR2 sequence corresponding to amino acid residues 50 to            56 of SEQ ID NO: 10; and        -   a CDR3 sequence corresponding to amino acid residues 89 to            97 of SEQ ID NO: 10;            -   wherein one or two of the amino acid residues in the                individual CDR sequences may be conservatively                substituted by a different amino acid.    -   31. The multispecific or bispecific antibody according to any        one of aspects 1 to 30 which is humanized.    -   32. A pharmaceutical composition comprising a first monospecific        antibody and a second monospecific antibody, wherein the first        monospecific antibody is capable of specifically binding to an        epitope within tissue factor pathway inhibitor (TFPI) amino acid        residues 1 to 181 of SEQ ID NO: 1;        -   and the second monospecific antibody is capable of            specifically binding to an epitope within TFPI amino acid            residues 182 to 276 of SEQ ID NO: 1.    -   33. The pharmaceutical composition according to aspect 32,        wherein the epitope of the first antibody comprises an amino        acid residue within the KPI-1 domain of tissue factor pathway        inhibitor (TFPI) or within the KPI-2 domain of TFPI and wherein        the epitope of the second antibody comprises an amino acid        residue within the KPI-3 domain of TFPI.    -   34. The pharmaceutical composition according to aspect 33,        wherein the first monospecific antibody comprises a heavy chain        (HC) comprising:    -   a CDR3 sequence corresponding to amino acid residues 98 to 110        of SEQ ID NO: 26;    -   and a light chain (LC) comprising:    -   a CDR3 sequence corresponding to amino acid residues 89 to 96 of        SEQ ID NO: 27;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   35. The pharmaceutical composition according to aspect 33,        wherein the second monospecific antibody comprises a heavy chain        (HC) comprising:        -   a CDR3 sequence corresponding to amino acid residues 99 to            107 of SEQ ID NO: 9;    -   and a light chain (LC) comprising:        -   a CDR3 sequence corresponding to amino acid residues 89 to            97 of SEQ ID NO: 10;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   36. The pharmaceutical composition according to aspect 35,        wherein the second monospecific antibody comprises a heavy chain        (HC) further comprising:        -   a CDR1 sequence corresponding to amino acid residues 31 to            35 of SEQ ID NO: 9; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            66 of SEQ ID NO: 9;    -   and the second monospecific antibody comprises a light chain        (LC) further comprising        -   a CDR1 sequence corresponding to amino acid residues 24 to            34 of SEQ ID NO: 10; and        -   a CDR2 sequence corresponding to amino acid residues 50 to            56 of SEQ ID NO: 10;    -   wherein one or two of the amino acid residues in the individual        CDR sequences may be conservatively substituted with a different        amino acid.    -   37. A multispecific or bispecific antibody or a pharmaceutical        composition comprising monospecific antibodies that compete with        the multispecific or bispecific antibody or pharmaceutical        composition comprising a combination of monospecific antibodies        according to any one of the former aspects.    -   38. A pharmaceutical composition comprising the multispecific or        bispecific antibody according to any one of aspects 1 to 31.    -   39. The multispecific or bispecific antibody according to any        one of aspects 1 to 31 or 37 or the pharmaceutical composition        comprising a combination of monospecific antibodies according to        any one of aspects 32 to 37 for use as a medicament.    -   40. The multispecific or bispecific antibody according to any        one of aspects 1 to 19 or 37 or the pharmaceutical composition        comprising a combination of monospecific antibodies according to        any one of aspects 32 or 37 for use in the treatment of        coagulopathy.    -   41. A eukaryotic cell which expresses the bispecific antibody        according to any one of aspects 1 to 31 or a fragment thereof.    -   42. Use of the bispecific antibodies according any one of        aspects 6 to 31 for measuring TFPIα levels in vitro.    -   43. A bispecific antibody according to any one of aspects 1 to        37 which selectively targets circulating TFPIα.

EXAMPLES Example 1: Immunisation, Fusion and Screening

RBF mice were immunised with human TFPIα (SEQ ID NO: 1) or a TFPIfragment, e.g. corresponding to residues 1-161 of SEQ ID NO: 1. Micewere injected subcutaneously: 20 μg human TFPI was mixed with completeFreund's adjuvant for the first injection. For subsequent immunisations,incomplete Freund's adjuvant was used with the same concentration of theantigen. Ten days after the final immunisation, eye-blood from mice wasscreened, using ELISA, for human TFPI specific antibodies. Mice withpositive serum titres were boosted with 10 μg of human TFPIα or TFPI(1-161) by intravenous injection and sacrificed after three days. Thespleens were removed aseptically and dispersed to a single cellsuspension. Fusion of spleen cells and myeloma cells was done by meansof the PEG-method or by electrofusion. The resulting hybridoma cellswere cloned by limiting dilution into microtiter plates. Supernatantsfrom individual hybridomas were initially screened by ELISA forexpression of antibodies capable of binding to full-length TFPIα or TFPIfragments.

Antibodies were purified from supernatants by standard protein Aaffinity chromatography and used to determine binding and affinity tohuman TFPI and TFPI neutralising activity in plasma (dilute prothrombintime). Hybridomas producing antibodies of interest were subcloned bylimited dilution and the original antibody profile was verified formaterial from subcloned hybridomas. Cells from subcloned hybridomas wereused for isolation of RNA and subsequent antibody cloning and sequenceidentification.

Example 2: Cloning and Sequencing of Mouse Anti-TFPI KPI-3/C-TerminalSpecific mAbs TFPI4F110, TFPI22F66 and TFPI22F71

The murine heavy chain and light chain sequences of TFPI antibodies:TFPI4F110, TFPI22F66 and TFPI22F71 were cloned and sequenced asdescribed in WO2012/001087.

Total RNA was extracted from hybridoma cells using the RNeasy-Mini Kitfrom Qiagen and used as a template for cDNA synthesis. cDNA wassynthesised in a 5′-RACE reaction using the SMART™ RACE cDNAamplification kit from Clontech. Subsequent target amplification of HCand LC sequences was performed by PCR using Phusion Hot Start polymerase(Finnzymes) and the universal primer mix (UPM) included in the SMART™RACE kit as forward primer. A reverse primer with the following sequencewas used for HC (VH domain) amplification:

(SEQ ID NO: 17) 5′-CTTGCCATTGAGCCAGTCCTGGTGCATGATGG-3′

A reverse primer with the following sequence was used for TFPI22F66 andTFPI22F71 LC (VL domain) amplification:

(SEQ ID NO: 18) 5′-GTTGTTCAAGAAGCACACGACTG-3′

A reverse primer with the following sequence was used for TFPI4F110 LCamplification:

(SEQ ID NO: 19) 5′-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3′

PCR products were separated by gel electrophoresis, extracted using theGFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciencesand cloned for sequencing using a Zero Blunt TOPO PCR Cloning Kit andchemically competent TOP10 E. coli (Invitrogen). Colony PCR wasperformed on selected colonies using an AmpliTaq Gold Master Mix fromApplied Biosystems and M13uni/M13rev primers. Colony PCR clean-up wasperformed using the ExoSAP-IT enzyme mix (USB). Sequencing was performedat Eurofins MWG Operon, Germany using M13uni(−21)/M13rev(−29) sequencingprimers. Sequences were analyzed and annotated using the VectorNTIprogram. All kits and reagents were used according to the manufacturer'sinstructions.

A single unique murine kappa type LC and a single unique murine HC,subclass IgG1 was identified for each of the hybridomas: TFPI4F110,TFPI22F66 and TFPI22F71. Amino acid sequences are provided in thesequence list: the leader peptide sequences are not included.

Example 3: Cloning and Sequencing of Mouse Anti-TFPI KPI-3/C-TerminalSpecific mAb 22F74, 41F41, 41F30, 22F132, 22F79 and Cloning andSequencing of Mouse Anti-TFPI KPI-1 mAb 2F3, 2F22, 2F45, 1F91 and 2F35

The murine heavy chain and light chain sequences of anti-TFPI antibodieswere cloned and sequenced as follows.

Total RNA was extracted from hybridoma cells using the RNeasy-Mini Kitfrom Qiagen and used as template for cDNA synthesis. cDNA wassynthesised in a 5′-RACE reaction using either the SMART™ or SMARTer™RACE cDNA amplification kit from Clontech. Subsequent targetamplification of HC and LC sequences was performed by PCR using PhusionHot Start polymerase (Finnzymes) and the universal primer mix (UPM)included in the SMART™/SMARTer™ RACE kit as forward primer. A reverseprimer with the following sequence was used for HC (VH domain)amplification:

(SEQ ID NO: 20) 5′-CCCTTGACCAGGCATCCCAG-3′

A reverse primer with the following sequence was used for LCamplification:

(SEQ ID NO: 21) 5′-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3′

PCR products were separated by gel electrophoresis, extracted using theGFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciencesand cloned for sequencing using a Zero Blunt TOPO PCR Cloning Kit andchemically competent TOP10 E. coli (Invitrogen). Colony PCR wasperformed on selected colonies using an AmpliTaq Gold Master Mix fromApplied Biosystems and M13uni/M13rev primers. Colony PCR clean-up wasperformed using the ExoSAP-IT enzyme mix (USB). Sequencing was performedat Eurofins MWG Operon, Germany using M13uni(−21)/M13rev(−29) sequencingprimers. Sequences were analyzed and annotated using the VectorNTIprogram. All kits and reagents were used according to the manufacturer'sinstructions.

A single unique murine kappa type LC and a single unique murine IgG HC,subclass was identified for each hybridoma. Amino acid sequences areprovided in the sequence list: the leader peptide sequences are notincluded.

Example 4: Cloning and Engineering of mAb 2021 and mAb 2021 Variants

This example describes cloning and engineering of anti-TFPI antibody:mAb2021 and variants thereof.

Total RNA was extracted from M-hTFPI 4F36A1B2 hybridoma cells using theRNeasy-Mini Kit from Qiagen and used as template for cDNA synthesis.cDNA was synthesized in a 5′-RACE reaction using the SMART™ RACE cDNAamplification kit from Clontech. Subsequent target amplification of HCand LC sequences was performed by PCR using Phusion Hot Start polymerase(Finnzymes) and the universal primer mix (UPM) included in the SMART™RACE kit as forward primer. A reverse primer with the following sequencewas used for HC (VH domain) amplification:

(SEQ ID NO: 20) 5′-CCCTTGACCAGGCATCCCAG-3′

A reverse primer with the following sequence was used for LCamplification:

(SEQ ID NO: 21) 5′-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3′

PCR products were separated by gel electrophoresis, extracted using theGFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciencesand cloned for sequencing using a Zero Blunt TOPO PCR Cloning Kit andchemically competent TOP10 E. coli (Invitrogen). Colony PCR wasperformed on selected colonies using an AmpliTaq Gold Master Mix fromApplied Biosystems and M13uni/M13rev primers. Colony PCR clean-up wasperformed using the ExoSAP-IT enzyme mix (USB). Sequencing was performedat Eurofins MWG Operon, Germany using either M13uni(−21)/M13rev(−29) orT3/T7 sequencing primers. Sequences were analyzed and annotated usingthe VectorNTI program. All kits and reagents were used according to themanufacturer's instructions.

A single unique murine kappa type LC and a single unique murine HC,subclass IgG1 was identified.

Generation of Expression Vectors for the Grafted Anti-Hz TFPI4F36A1B2

A series of CMV promotor-based based expression vectors (pTT vectors)were generated for transient expression of anti-TFPI antibody/antibodyfragment in the HEK293-6E EBNA-based expression system developed by YvesDurocher (Durocher et al. Nucleic Acid Research, 2002). In addition tothe CMV promotor, the pTT-based vectors contain a pMB1 origin, an EBVorigin and the Amp resistance gene.

Based on the cloned murine anti-TFPI4F36 Å1 B2 VH and VL sequences ahumanized version of anti-TFPI4F36 was designed by CDR grafting on humangermline sequences. DNA sequences for grafted HzTFPI4F36 VH and VLregions were synthesized (GENEART AG) according to the humanizationdesign of the antibody described above. The sequences were obtained withthe basic minimal CDR grafting and no additional back mutations. Therespective LC and HC germline leader peptide sequences were include inthe constructs as well as a Kozak sequence (5′-GCCGCCACC-3′) immediatelyupstream of the ATG start codon.

pTT-based expression vectors were generated for transient expression ofthe grafted TFPI4F36 antibody as a human kappa/IgG4(S241P) isotype. Theproline mutation at position 241 (numbering according to Kabat,corresponding to residue 228 per the EU numbering system (Edelman G. M.et AL., Proc. Natl. Acad. USA 63, 78-85 (1969)) was introduced in theIgG4 hinge region to eliminated formation of monomeric antibodyfragments, i.e. “half-antibodies” comprised of one LC and one HC.

The VH fragment was excised from the GENEART cloning vector and clonedinto a linearized pTT-based vector containing the sequence for a humanIgG4(S241P) CH domain and subsequently transformed into E. coli forselection. The sequence of the final construct was verified by DNAsequencing. The VL fragment was excised from the GENEART cloning vectorand cloned into a linearized pTT-based vector containing the sequencefor a human kappa CL domain and subsequently transformed into E. colifor selection. The sequence of the final constructs was verified by DNAsequencing.

Site-Directed Mutagenesis to Isolate mAb 2021

A series of human-to-mouse reverse mutation (referred to as backmutations) were generated in the light chain (LC) and heavy chain (HC)of the grafted HzTFPI4F36.

Site-directed mutagenesis was performed to introduce human-to-mousereverse mutations (henceforth referred to as back mutations) at thespecific residues in the grafted HzTFPI4F36 LC/HC constructs to optimizethe grafted constructs. Mutations were introduced by two differentmethods:

1. QuikChange® Site-Directed or Multi Site-Directed Mutagenesis kitsfrom Stratagene were used to introduce point mutations and combinationmutations. The kits were used according to the manufacturer's protocol.

2. Standard 2-step overlapping PCR methods were also used to introducepoint mutations and to generate combination mutations.

The LC and HC expression plasmids for grafted HzTFPI4F36 were used astemplates for the mutagenesis. The sequences of all final constructswere verified by DNA sequencing.

The final sequences for mAb 2021 HC carries a FR2 region with 4 backmutations rendering the framework sequence identical to original mouseFR2 and 3 CDR2 mutants, i.e. a total of 7 HC back mutations (A40T, G42E,G44R, S49A, Y59F, A60P, K64Q) compared to the original grafted sequence(numbering according to Kabat). The LC sequence is the graftedHzTFPI4F36 LC sequence. CDRs and frameworks defined according to Kabat.

The amino acid sequences for the HC and LC of the final mAb 2021 arelisted as SEQ ID NO: 50 and SEQ ID NO: 51, respectively.

In order to improve the expression yield the original signal peptidesequences (human germline sequence) for both HC and LC, were exchangedfor the human CD33 signal peptide (SP). The signal peptide sequenceswere exchanged by standard PCR amplification of the HC or LC fragmentwith primers containing a Kozak element (GCCGCCACC), start codon and theCD33 signal sequence (sense primer) and stop codon and EcoRI restrictionsite (anti-sense primer). The amplified fragments were cloned intolinearized pTT-based vectors and transformed into E. coli for selection.The sequence of the final construct was verified by DNA sequencing.

Generation of Lower Affinity Variants of mAb 2021

During humanization of anti-TFPI4F36A1B2 to obtain mAb 2021 a number oflower affinity variants were identified. The variants were generated asdescribed above by site-directed mutagenesis. mAb 2007 and mAb 2014 weretwo such lower affinity variants. mAb 2007 carries a single A60P backmutations in the VH domain compared to the sequence for the graftedHzTFPI4F36 (numbering according to Kabat). Compared to the sequence forthe grafted HzTFPI4F36, mAb 2014 carries a FR2 region with 4 backmutations (Y36L, K39R, Q42E, Q45K) in the VL domain, rendering theframework sequence identical to original mouse FR2 (numbering accordingto Kabat). The heavy chain of mAb 2014 corresponds to the graftedHzTFPI4F36. The two variants have TFPI binding affinities that are lowerby at least one or two orders of magnitude, respectively compared to mAb2021. The initial grafted HzTFPI4F36 variant (mAb 2000) exhibited TFPIbinding affinities approximately three orders of magnitude lowercompared to mAb 2021.

Expression vectors for expression of Fab fragments of mAb 2021 and mAb2021 variants were generated as described in example 6. The IgG4-basedHC was truncated in the hinge region after the cysteine corresponding toposition 227 in the HC sequence for mAb 2021, SEQ. ID. NO: 50. Thetruncation leaves the C-terminal cysteine available for chemicalconjugation. The Fab fragment of mAb 2007 was expressed as Fab 0094,using the mAb 2021 LC vector and the truncated mAb 2007 HC vectordescribed above. The Fab fragment of mAb 2014 was expressed as Fab 0095,using the mAb 2014 LC vector and the truncated mAb 2014 HC vectordescribed above. The Fab fragment of mAb 2000 was expressed as Fab 0313,using the mAb 2021 LC vector and the truncated HC for the graftedHzTFPI4F36.

Example 5: Generation of Expression Vectors for Mouse/Human ChimericTFPI4F110, TFPI22F66, TFPI22F71 TFPI2F22 mAbs

A series of CMV promoter-based expression vectors (pTT vectors) weregenerated for transient expression of a mouse/human chimeric anti-TFPIantibody/antibody fragment in the HEK293-6E EBNA-based expression systemdeveloped by Yves Durocher (Durocher et al. Nucleic Acid Research, 2002)or the EXPI293F system from Invitrogen. In addition to the CMV promoter,the pTT-based vectors contain a pMB1 origin, an EBV origin and the Ampresistance gene. The anti-TFPI antibody variants including Fab fragmentswere expressed transiently in HEK293-6E or EXPI293F cells byco-transfection of LC and HC expression vectors as described in example7. pTT-based LC expression vectors were generated for transientexpression of chimeric anti-TFPI mAb and Fab fragments. Initially, theregion corresponding to the VL domains of either TFPI4F110, TFPI22F66,TFPI22F71 or TFPI 2F22 were PCR amplified in a 2-step reaction from anoriginal TOPO sequencing clone, using primers specific for the N andC-terminal sequences. The original murine signal peptide was exchangedfor the human CD33 signal peptide by 2-step overlapping PCR. The primarysense primers carries the C-terminal part of the CD33 signal peptidesequence and the secondary sense primer contained a HindIII restrictionsite for cloning purposes, a Kozak sequence (5′-GCCGCCACC-3′)immediately upstream of the ATG start codon and the N-terminal part ofthe CD33 signal peptide sequences. The anti-sense primer contained anin-frame BsiWI restriction site in the VL/CL transition sequence.

The amplified fragments were cloned into a linearised pTT-based vectorcontaining the sequence for a human kappa CL domain and subsequentlytransformed into E. coli for selection. The sequence of the finalconstruct was verified by DNA sequencing.

pTT-based HC expression vectors were generated for transient expressionof chimeric anti-TFPI mAbs. Initially, the region corresponding to theVH domains of either TFPI4F110, TFPI22F66, TFPI22F71 or TFPI2F22 werePCR amplified in a 2-step reaction from an original TOPO sequencingclone, using primers specific for the N- and C-terminal sequences. Theoriginal murine signal peptide was exchanged for the human CD33 signalpeptide by 2-step overlapping PCR. The primary sense primers carries theC-terminal part of the CD33 signal peptide sequence and the secondarysense primer contained a HindIII restriction site for cloning purposes,a Kozak sequence (5′-GCCGCCACC-3′) immediately upstream of the ATG startcodon and the N-terminal part of the CD33 signal peptide sequences. Theanti-sense primer contained an in-frame NheI restriction site at theVH/CH transition.

The generated VH domain PCR fragment was restriction digested and clonedinto a linearised pTT-based vector containing the sequence of anengineered human IgG4(S241 P) CH domain and subsequently transformedinto E. coli for selection. The proline mutation at position 241(numbering according to Kabat, corresponding to residue 228 per the EUnumbering system (Edelman G. M. et al., Proc. Natl. Acad. USA 63: 78-85(1969))) in the IgG4 hinge region was introduced to eliminated formationof monomeric antibody fragments, i.e. “half-antibodies” comprised of oneLC and one HC. The sequence of the final construct was verified by DNAsequencing.

The chimeric version of TFPI4F110 was expressed recombinantly as mAb4F110, using the TFPI4F110 LC and HC vectors.

The chimeric version of TFPI22F66 was expressed recombinantly as mAb22F66, using the TFPI22F66 LC and HC vectors.

The chimeric version of TFPI22F71 was expressed recombinantly as mAb22F71, using the TFPI22F71 LC and HC vectors.

The chimeric version of TFPI2F22 was expressed recombinantly as mAb2F22, using the TFPI2F22 LC and HC vectors.

Example 6: Fab Components for Bispecific Molecules

To generate Fab fragments suitable for Fab-Fab chemical conjugates, anexpression vector for expression of a truncated mAb 4F110 HC wasgenerated. The IgG4-based HC of mAb 4F110 was truncated in whatcorresponds to the hinge region after the cysteine in position 227 inthe human IgG4 constant region (numbering according to SEQ ID NO: 50).The truncation leaves the C-terminal cysteine available for chemicalconjugation. The truncated sequence was generated by introducing a stopcodon after the cysteine residue by site-directed mutagenesis using theQuikChange® Site-Directed mutagenesis kit from Stratagene. The sequencesof the final constructs were verified by DNA sequencing. The Fabfragment of mAb 4F110 was expressed as Fab 0089, using the mAb 4F110 LCvector and the truncated HC vector described above (SEQ ID NO: 9 and SEQID NO: 10).

For Fab-Fab chemical conjugates, an expression vector for expression ofa truncated mAb 2021 HC was also generated. Cloning, humanisation andexpression of mAb 2021 is described in WO2010/072691, which is herebyincorporated by reference. The IgG4-based HC of mAb 2021 was truncatedin the hinge region after the cysteine in position 227 in the human IgG4constant region of mAb 2021 HC, SEQ ID no: 50. The truncation leaves theC-terminal cysteine available for chemical conjugation. The truncatedsequence was generated by introducing a stop codon after the cysteineresidue by site-directed mutagenesis using the QuikChange® Site-Directedmutagenesis kit from Stratagene. The sequences of all final constructswere verified by DNA sequencing. The Fab fragment of mAb2021 wasexpressed as Fab 0088, using the mAb 2021 LC vector and the truncated HCvector described above (SEQ ID NO: 3 and SEQ ID NO: 4).

Heavy chain expression vectors for expression of lower affinity variantsof mAb 2021 were also generated. The mAb 2021 variants originate fromthe humanisation of mAb 2021 and are described in WO2010/072691. TheIgG4-based HCs were truncated in the hinge region after the cysteinecorresponding to position 227 in the human IgG4 constant region of mAb2021 HC, SEQ ID no: 50. The truncation leaves the C-terminal cysteineavailable for chemical conjugation. To assemble the expression vectors,the region corresponding to the VH domain of the HCs of mAb 2000,mAb2007 and mAb2014 described in example 4 were cloned using standardrestriction enzyme based cloning into a linearised pTT-based toolboxvector containing the sequence of the truncated human IgG4 CH domain.The IgG4-based HCs were truncated in the hinge region after the cysteinein position 227 as described above. The sequences of the finalconstructs were verified by DNA sequencing. The lower affinity Fabfragments of mAb2007, mAb 20014 and mAb 2000 were expressed as Fab 0094(SEQ ID NO: 5 and SEQ ID NO: 6), Fab 0095 (SEQ ID NO: 7 and SEQ ID NO:8) and Fab 0313 (SEQ ID NO: 58 and SEQ ID NO: 59), respectively.

The above described method for generation of truncated HC expressionvectors for Fab 0094,0095, 0313 was also used in the generation of otherFab fragments of the present invention such as Fab 2F22 (SEQ ID NO: 40and SEQ ID NO: 41).

Example 7: Expression and Purification of mAbs and Fabs

The anti-TFPI antibody variants including Fab fragments were expressedtransiently in suspension cultures of either HEK293-6E cells or EXPI293Fcells, by co-transfection of LC and HC expression vectors. The followingprocedures describes the generic transfection protocol used forsuspension adapted HEK293-6E cells or EXPI293F cells.

HEK293-6E Protocol

HEK293-6E cells were grown in suspension in FreeStyle™ 293 expressionmedium (Gibco) supplemented with 25 μg/ml Geneticin (Gibco), 0.1% v/v ofthe surfactant Pluronic F-68 (Gibco) & 1% v/v Penicillin-Streptomycin(Gibco). Cells were cultured in Erlenmeyer shaker flasks in shakerincubators at 37° C., 8% CO₂ and 125 rpm and maintained at celldensities between 0.1-1.5×10⁶ cells/ml.

HEK293-6E Transfection

-   -   The cell density of cultures used for transfection was        0.9-2.0×10⁶ cells/ml.    -   A mix of 0.5 μg LC vector DNA+0.5 μg HC vector DNA was used per        ml cell culture.    -   The DNA was diluted in Opti-MEM media (Gibco) 30 μl media/μg        DNA, mixed and incubated at room temperature (23-25° C.) for 5        min.    -   293Fectin™ (Invitrogen) was used as transfection reagent at a        concentration of 1 μl per μg DNA.    -   The 293Fectin™ was diluted 30× in Opti-MEM media (Gibco), mixed        and incubated at room temperature (23-25° C.) for 5 min.    -   The DNA and 293Fectin solutions were mixed and left to incubate        at room temperature (23-25° C.) for 25 min.    -   The DNA-293Fectin mix was then added directly to the cell        culture.    -   The transfected cell culture was transferred to a shaker        incubator at 37° C., 8% CO₂ and 125 rpm.    -   3-6 days post transfection, cell culture supernatants were        harvested by centrifugation, followed by filtration through a        0.22 μm PES filter (Corning).    -   Quantitative analysis of antibody production was performed by        Biolayer Interferometry directly on clarified cell culture        supernatants using the FortéBio Octet system and protein A        biosensors or quantitative protein A HPLC.        EXPI293F Protocol

EXPI293F cells were grown in suspension in Expi293™ expression medium(Life Technologies). Cells were cultured in Erlenmeyer shaker flasks inan orbital shaker incubator at 36.5° C., 8% CO₂ and 85-125 rpm andmaintained at cell densities between 0.4-4×10⁶ cells/ml.

EXPI293F Transfection

-   1) Separate dilutions of DNA and transfection reagent are initially    prepared.    -   a) Use a total of 1 μg of vector DNA (0.5 ug LC vector and 0.5        ug HC vector) per ml cell culture. Dilute the DNA in Opti-MEM        media (Gibco) 50 μl medium/μg DNA, mix and incubate at room        temperature (23-25° C.) for 5 min.    -   b) Use Expifectamin™ 293 (Life Technologies) as transfection        reagent at a concentration of 2.7 μl per μg DNA. Dilute the        Expifectamin™ solution 18.5× in Opti-MEM media (Gibco), mix and        incubate at room temperature (23-25° C.) for 5 min.-   2) Mix DNA and Expifectamin™ 293 dilutions and leave to incubate at    room temperature (23-25° C.) for 10 min.-   3) Add the DNA-Expifectamin™ 293 mix directly to the EXPI293F cell    culture.    -   a) At the time of transfection the cell density of the EXPI293F        culture should be 2.8-3.2×10⁶ cells/ml.    -   4) Transfer the transfected cell culture to an orbital shaker        incubator at 36.5° C., 8% CO₂ and 85-125 rpm.    -   5) 18 hrs post transfection, add 5 ul Expifectamin™ 293        Transfection Enhancer 1/ml culture and 50 ul Expifectamin™ 293        Transfection Enhancer 2/ml culture and return culture to an        orbital shaker incubator at 36.5° C., 8% CO₂ and 85-125 rpm.    -   6) 5 days post transfection, cell culture supernatants were        harvested by centrifugation, followed by filtration through a        0.22 μm PES filter unit (Corning).        General Purification Protocol

mAb variants were purified by standard affinity chromatography usingMabSelectSuRe resin from GE Healthcare according to manufacturer'sinstructions. The purified antibodies were buffer exchanged to PBSbuffer pH7.2.

Fab fragments were purified by standard affinity chromatography usingKappaSelect resin developed by GE Healthcare. The purified Fab fragmentswere buffer exchanged to PBS buffer pH7.2.

Quality assessment and concentration determination was done by SEC-HPLC.

Example 8: Conjugation of Fab 0088 and Fab 0089 with1,8-N,N′-bis(maleimido)-3,6-dioxaoctane (ref. 9000)

Fab 0088 (Fab fragment of mAb 2021) (5 mg, 3.92 mg/ml, 0.104 mmol) inPBS buffer was mixed with a solution of Tris(3-sulfonatophenyl)phosphinehydrate sodium salt (3.2 ml, 10 mg/ml, technical grade, (Alfa Aesar#39538, CAS: 63995-70-0). The reaction was left for 1 hour and 30minutes at ambient temperature. The mixture was subjected to bufferexchange into 50 mM acetate buffer (dilute acetic acid adjusted to pH 5with sodium hydroxide; buffer A) using an Amicon Ultra centrifugalfilter device (5 kDa MWCO, Millipore). The protein was immobilised on apreconditioned HiTrap SP HP column (GE Healthcare) and eluded with agradient in which buffer B was buffer A containing 300 mM sodiumchloride.

The eluded protein (3 ml, 0.6 mg) was mixed with a solution of1,8-N,N′-bis(maleimido)-3,6-dioxaoctane (Thermo Scientific/Pierce,product no. 22336; 6.2 mg in 2 ml buffer). The resulting mixture wasincubated at ambient temperature for 2.5 hours. The mixture wassubjected to buffer exchange into 50 mM acetate buffer (dilute aceticacid adjusted to pH 5 with sodium hydroxide; buffer A) using an AmiconUltra centrifugal filter device (5 kDa MWCO, Millipore). The protein wasimmobilised on a preconditioned HiTrap SP HP column (GE Healthcare) andeluded with a gradient in which buffer B was buffer A containing 300 mMsodium chloride.

Fab 0089 (Fab fragment of mAb 4F110) (5 mg, 3.58 mg/ml, 0.105 mmol) inPBS buffer was mixed with a solution of Tris(3-sulfonatophenyl)phosphinehydrate sodium salt (3.2 ml, 10 mg/ml, technical grade, (Alfa Aesar#39538, CAS: 63995-70-0).

The reaction was left for 2 hours and 15 minutes at ambient temperature.The mixture was subjected to buffer exchange into 50 mM acetate buffer(dilute acetic acid adjusted to pH 5 with sodium hydroxide; buffer A)using an Amicon Ultra centrifugal filter device (5 kDa MWCO, Millipore).The protein was immobilised on a preconditioned HiTrap SP HP column (GEHealthcare) and eluded with a gradient in which buffer B was buffer Acontaining 300 mM sodium chloride.

The two solutions (Fab 0088 reduced and coupled to linker, 4 ml; and Fab0089, reduced, 4 ml) were mixed. The resulting mixture was left atambient temperature overnight. The solution was concentrated and loadedonto a Superdex 200 10/300 GL column (GE Healthcare) using an Äkta FPLCinstrument and eluded in buffer (9.7 mM Histidin, 2.3 mM CaCl₂, 308 mMNaCl, 8.8 mM Sucrose, 0.01% Tween 80, pH 7.0). The isolated fractionswere analysed by SDS-PAGE analysis, analytical size exclusionchromatography, and Edman sequence determination.

Example 9: Preparation of1,16-N,N′-bis(maleimido)-4,13-diaza-7,10-dioxa-3,14-dioxohexadecane

N-Maleoyl-β-alanine (1.26 g, 7.4 mmol) was dissolved in DCM (20 ml).N,N′-diisopropylcarbodiimide (1.15 ml, 7.4 mmol) was added. The mixturewas stirred for 30 minutes. A solution of 1,8-diamino-3,6-dioxaoctane inDCM was added dropwise to the stirring solution over a period of 60minutes. The mixture was stirred for 1 h. The solution was concentratedin vacuo to dryness. The crude solids were suspended in water/MeCN (2:1)with some acetic acid present. The mixture was purified using RP HPLC(0-50% MeCN in water, 0.1% TFA, C₁₈ column). The product wascharacterised by LCMS (ESI): 451 ([M+H]⁺:) and NMR.

Example 10: Conjugation of Fab 0088 and Fab 0089 with1,16-N,N′-bis(maleimido)-4,13-diaza-7,10-dioxa-3,14-dioxohexadecane(ref. 9002)

Fab 0089 (5 mg, 3.58 mg/ml, 1.42 ml, 104 nmol) was buffer exchanged into25 mM sodium phosphate buffer, pH 7. The final volume was 2000microliter (50 micromolar, 2.5 mg/ml).Bis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt from asolution in the phosphate buffer was added to an end-concentration of200 micromolar (Bis(p-sulfonatophenyl)phenylphosphine dipotassiumdihydrate salt, CAS: 151888-20-9, Sigma-Aldrich; 2.24 mg, 4.19 micromol)was dissolved in 1 ml 25 mM sodium phosphate buffer, pH 7 resulting in aconcentration of 4.19 mM (dilution factor: 20.9); 95 microliter wasadded to the protein solutions. (2000 microliter total volume).

1,16-N,N′-bis(maleimido)-4,13-diaza-7,10-dioxa-3,14-dioxohexadecane (5mg, 11 micromol) was added to the solution. The mixture was incubated atambient temperature for 45 minutes.

The sample was diluted with 5 mM acetate buffer, pH 5. The sample wasloaded onto a HiTrap SP HP column (1 ml). The column was washed with 5mM acetate and 25 mM acetate buffer. The compound was eluded with 25 mMacetate buffer containing 1 M NaCl.

Fab 0088 (5 mg, 3.92 mg/ml, 1.27 ml, 104 nmol) was buffer exchanged into25 mM sodium phosphate buffer, pH 7. The final volume was 2000microliter (50 micromolar, 2.5 mg/ml).Bis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt from asolution in the phosphate buffer was added to an end-concentration of200 micromolar (Bis(p-sulfonatophenyl)phenylphosphine dipotassiumdihydrate salt, CAS: 151888-20-9, Sigma-Aldrich; 2.24 mg, 4.19 micromol)was dissolved in 1 ml 25 mM sodium phosphate buffer, pH 7 resulting in aconcentration of 4.19 mM (dilution factor: 20.9); 95 microliter wasadded to the protein solutions (2000 microliter total volume).

The two protein solutions were mixed, buffer exchanged to 25 mM acetatebuffer, pH 5, and concentrated to half the volume. The mixture wasincubated at ambient temperature for 3 hours. The solution was loadedonto a Superdex 200 10/300 GL column that had been preconditioned in 10mM L-Histidine, 150 mM NaCl, pH 7. The compound was eluded using saidbuffer. The selected fractions were pooled and analysed using SDS-PAGEanalysis, LCMS (m/z: 96144, [M-NH₃+H]⁺), Reversed phase HPLC, and Edmansequence determination. This conjugate was designated 9002.

Example 11: Conjugation of Fab 2F22 and Fab 0089 with1,16-N,N′-bis(maleimido)-4,13-diaza-7,10-dioxa-3,14-dioxohexadecane(ref. 9028)

In complete analogy with the example above, Fab 2F22 (3.9 mg) and Fab0089 (8.0 mg) were buffer-exchanged to phosphate buffered saline usingan Amicon Ultra centrifugal device (Millipore Corp., 10 kDa MWCO). Theconcentrations of both proteins were adjusted to 50 micromolar.

Bis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt from asolution in the phosphate buffer was added to an end-concentration of200 micromolar. The resulting mixtures were incubated over night at roomtemperature. Fab 2F22 was buffer-exchanged into 15 mM sodium acetatebuffer, 1 M sodium chloride, pH 5.0.

Fab 0089 was buffer-exchanged into 15 mM sodium acetate buffer, pH 4.5.1,16-N,N′-bis(maleimido)-4,13-diaza-7,10-dioxa-3,14-dioxohexadecane (tipof a spatula) was added to the solution. The resulting mixture was keptat room temperature for 30 minutes. The mixture was loaded onto apreconditioned HiTrap SP HP column (GEHealthcare, 1 ml). The immobilisedprotein was washed with 15 mM sodium acetate buffer, pH 4.5, 25 CV. Theprotein was eluded from the column with 15 mM sodium acetate buffer, 1 MNaCl, pH 5.0 and mixed with Fab 2F22. The resulting mixture wasconcentrated in Amicon Ultra centrifugal spin filter (MWCO 10 kDa). Endvolume: 400 microliter. The mixture was incubated for 20 h. The samplewas injected on gel-permeation column that had been pre-conditioned in 5mM HEPES, 150 mM NaCl, pH 7.3 (HiLoad16/600 Superdex 200 column, GEHeathcare). Using a flow of 1 ml/min of 5 mM HEPES, 150 mM NaCl, pH 7.3in water, the sample was eluded from the column. The fractionscontaining the desired conjugate were identified using SDS-PAGEanalysis. The pooled fractions were concentrated to a volume of 1000microliter using an Amicon Ultra Centrifugal device (MWCO 10 kDa). LCMS(m/z: 95629, [M+H]⁺). This conjugate was designated 9028.

Example 12: Conjugation of Fab 0094 and Fab 0089 with1,16-N,N′-bis(maleimido)-4,13-diaza-7,10-dioxa-3,14-dioxohexadecane(ref. 9004)

In complete analogy with the example above, a conjugate of Fab 0094 andFab 0089 was formed. LCMS (m/z: 95994, [M+H]⁺). This conjugate wasdesignated 9004.

Example 13: Conjugation of Fab 0095 and Fab 0089 with1,16-N,N′-bis(maleimido)-4,13-diaza-7,10-dioxa-3,14-dioxohexadecane(ref. 9005)

In complete analogy with the example above, a conjugate of Fab 0095 andFab 0089 was formed. LCMS (m/z: 95928, [M-NH₃+H]⁺). This conjugate wasdesignated 9005.

Example 14: Preparation of Bis(bromoacetyl)ethylene diamine

Bromoacetic acid (2.1 g, 15 mmol) was dissolved in DCM (40 ml). Thesolution was added dropwise to a suspension of immobilised carbodiimideon PS resin (N-cyclohexylcarbodiimide-N′-methyl polystyrene;Novabiochem, prod#: 8.55029.0025; 2.3 mmol/g; 10 g) over a period of 15minutes. A solution of diamine (450 mg, 7.5 mmol) in DCM (20 ml) wasadded dropwise to the stirring solution over a period of 20 minutes. Themixture was stirred for 1 h. The solution was filtered and concentratedin vacuo to dryness. LCMS (m/z: 302.9, [M+H]⁺).

Example 15: Conjugation of Fab 0088 and Fab 0089 usingbis(bromoacetyl)ethylene diamine (ref. 9029)

Fab 0088 and Fab 0089 were buffer-exchanged to phosphate buffered salineusing an Amicon Ultra centrifugal device (Millipore Corp., 10 kDa MWCO).The concentrations of both proteins were adjusted to 50 micromolar.Bis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt from asolution in the phosphate buffer was added to an end-concentration of200 micromolar. The resulting mixtures were incubated over night at roomtemperature.

The proteins were buffer exchanged to 20 mM triethanolamin, 1 M NaCl,200 micromolar Bis(p-sulfonatophenyl)phenylphospine dihydratedipotassium salt, pH 8.5.Fab 0089 (60 mg, 10 mg/ml) was mixed withbis(bromoacetyl)ethylene diamine (37 mg).The mixture was incubated atambient temperature for 30 minutes. LCMS (m/z: 47858 [M-NH₃+H]⁺). Thesample was diluted (about 10-fold) with 15 mM acetate buffer, pH 4.5.The sample was loaded onto a pre-conditioned HiTrap SP HP column (5 ml).The column was washed with 15 mM acetate buffer. The compound was eludedwith an aqueous buffer of 20 mM triethanolamin and 1 M NaCl.pH 8.5. Thetwo protein solutions were mixed, and concentrated/buffer exchanged into20 mM triethanolamin, 1 M NaCl, pH 8.5, 200 micromolarBis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt, pH 8.5using an Amicon Ultra centrifugal device (Millipore Corp., 10 kDa MWCO).The mixture was incubated at ambient temperature for 24 hours. Thesolution was loaded onto a Superdex200 26/60 GL column that had beenpreconditioned in 15 mM HEPES, 150 mM NaCl, pH 7.3 (HiLoad26/60 Superdex200 column, GE Heathcare). Using a flow of 2.5 ml/min of in 15 mM HEPES,150 mM NaCl, pH 7.3 in water, the sample was eluded from the column. Thefractions containing the desired conjugate were identified usingSDS-PAGE analysis. The pooled fractions were concentrated to a volume of1000 microliter using an Amicon Ultra Centrifugal device (MWCO 10 kDa).LCMS (m/z: 95834, [M-NH₃+H]⁺).

Example 16: Conjugation of Fab 0094 and Fab 0089 usingbis(bromoacetyl)ethylene diamine (ref. 9030)

Fab 0094 and Fab 0089 were buffer-exchanged to phosphate buffered salineusing an Amicon Ultra centrifugal device (Millipore Corp., 10 kDa MWCO).The concentrations of both proteins were adjusted to 50 micromolar.Bis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt from asolution in the phosphate buffer was added to an end-concentration of200 micromolar. The resulting mixtures were incubated over night at roomtemperature. The proteins were buffer exchanged to 20 mM triethanolamin,1 M NaCl, 200 micromolarBis(p-sulfonatophenyl)phenylphospine dihydratedipotassium salt, pH 8.5.

Fab 0089 (60 mg, 10 mg/ml) was mixed with bis(bromoacetyl)ethylenediamine (37 mg).The mixture was incubated at ambient temperature for 30minutes. LCMS (m/z: 47858 [M-NH₃+H]⁺).

The sample was diluted (about 10-fold) with 15 mM acetate buffer, pH4.5. The sample was loaded onto a pre-conditioned HiTrap SP HP column (5ml). The column was washed with 15 mM acetate buffer. The compound waseluded with an aqueous buffer of 20 mM triethanolamin and 1 M NaCl.pH8.5.

The two protein solutions were mixed, and concentrated/buffer exchangedinto 20 mM triethanolamin, 1 M NaCl, pH 8.5, 200 micromolarBis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt, and 200micromolar sodium iodide, pH 8.5 using an Amicon Ultra centrifugaldevice (Millipore Corp., 10 kDa MWCO). The mixture was incubated atambient temperature for 24 hours. The solution was loaded onto aSuperdex200 26/60 GL column that had been preconditioned in 15 mM HEPES,150 mM NaCl, pH 7.3 (HiLoad26/60 Superdex 200 column, GE Heathcare).Using a flow of 2.5 ml/min of in 15 mM HEPES, 150 mM NaCl, pH 7.3 inwater, the sample was eluded from the column. The fractions containingthe desired conjugate were identified using SDS-PAGE analysis. Thepooled fractions were concentrated to a volume of 1000 microliter usingan Amicon Ultra Centrifugal device (MWCO 10 kDa). LCMS (m/z: 95664,[M-NH₃+H]⁺). This compound was designated 9030.

Example 17: Conjugation of Fab 0313 and Fab 0089 usingbis(bromoacetyl)ethylene diamine (ref. 9031)

Fab 0094 and Fab 0089 were buffer-exchanged to phosphate buffered salineusing an Amicon Ultra centrifugal device (Millipore Corp., 10 kDa MWCO).The concentrations of both proteins were adjusted to 50 micromolar.Bis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt from asolution in the phosphate buffer was added to an end-concentration of200 micromolar. The resulting mixtures were incubated over night at roomtemperature.

The proteins were buffer exchanged to 20 mM triethanolamin, 1 M NaCl,200 micromolarBis(p-sulfonatophenyl)phenylphospine dihydrate dipotassiumsalt, pH 8.5.

Fab 0089 (60 mg, 10 mg/ml) was mixed with bis(bromoacetyl)ethylenediamine (37 mg).The mixture was incubated at ambient temperature for 30minutes. LCMS (m/z: 47858 [M-NH₃+H]⁺).

The sample was diluted (about 10-fold) with 15 mM acetate buffer, pH4.5. The sample was loaded onto a pre-conditioned HiTrap SP HP column (5ml). The column was washed with 15 mM acetate buffer. The compound waseluded with an aqueous buffer of 20 mM triethanolamin and 1 M NaCl, pH8.5.

The two protein solutions were mixed, and concentrated/buffer exchangedinto 20 mM triethanolamin, 1 M NaCl, pH 8.5, 200 micromolarBis(p-sulfonatophenyl)phenylphospine dihydrate dipotassium salt, and 200micromolar sodium iodide, pH 8.5 using an Amicon Ultra centrifugaldevice (Millipore Corp., 10 kDa MWCO). The mixture was incubated atambient temperature for 24 hours. The solution was loaded onto aSuperdex200 26/60 GL column that had been preconditioned in 15 mM HEPES,150 mM NaCl, pH 7.3 (HiLoad26/60 Superdex 200 column, GE Heathcare).Using a flow of 2.5 ml/min of in 15 mM HEPES, 150 mM NaCl, pH 7.3 inwater, the sample was eluded from the column. The fractions containingthe desired conjugate were identified using SDS-PAGE analysis. Thepooled fractions were concentrated to a volume of 1000 microliter usingan Amicon Ultra Centrifugal device (MWCO 10 kDa). LCMS (m/z: 95638,[M-NH₃+H]⁺).

Example 18: Thrombin Generation Assay

The effect of antibodies on thrombin generation was studied in normalhuman plasma (NHP, HemosIL Calibration plasma) supplemented with 10 μMPS/PC: Phosphatidylserine/phosphatidylcholine 25/75%. Coagulation wasinitiated by re-calcification and addition of Innovin® (1.0 μM).Haemophilia A-like plasma was obtained by the addition of 100 μg/mlsheep anti human FVIII antibody (commercially available).

Thrombin activity was assessed continuously following the conversion ofthe fluorogenic substrate Z-Gly-Gly-Arg-AMC.HCl (I-1140), from Bachem(Bubendorf, Switzerland). Fluorescence was measured in a microtiterplate Fluorskan Ascent fluorometer (Thermo Labsystems, Helsinki,Finland) with excitation and emission wavelengths set at 368 and 460 nm,respectively. A calibrator was used to allow calculation of the amountof thrombin formed and correction of the obtained relative fluorescenceunits for inner-filter effects and fluorogenic substrate consumption. Inaddition, the contribution to substrate conversion bythrombin-α2-macroglobulin complexes was subtracted. These correctionswere performed automatically by means of the calibrated automatedthrombogram (CAT) computer software provided by Synapse BV (Maastricht,the Netherlands). The first derivative of the data was taken thatyielded the thrombin generation curve, allowing calculation of i) lagtime, ii) total area under the curve, the endogenous thrombin potential(ETP), iii) thrombin peak height (Peak), iv) time to peak (ttPeak) andv) maximal rate of thrombin generation (Rate).

Example 19: Binding Interaction Analysis

Binding interaction analysis was obtained by Surface Plasmon Resonancein a Biacore T200 instrument. Human Fab Binder (Human Fab Capture Kit,GE Healthcare) was immobilized to a series S CM4 sensor chip (GEHealthcare) in flow-cells 1-4 at 10000 response units (RU) using theamine coupling protocol available within the Biacore T-200 controlsoftware (GE Healthcare) and the reagents provided with the AmineCoupling Kit (GE Healthcare).The relevant Fab fragment or Fab-Fabconjugate was captured in flow-cell 2, 3 or 4 at a fixed concentration.Different concentrations of TFPI were tested by diluting TFPI in therunning buffer (10 mM Hepes, pH 7.4, 300 mM NaCl, 5 mM CaCl₂, 0.05%Surfactant P20, 1 mg/ml BSA). Each sample was assayed using 300 secondsof contact time followed by 600 seconds of dissociation time at 50μl/min flow rate. A buffer blank was also assayed. The sensor surfacewas regenerated with 10 mM Glycine pH 2.1 with two cycles each of 30seconds at 50 μl/min flow rate.

Biacore T200 Evaluation software was used to analyse the data.Determination of binding constants (k_(on), k_(off), K_(D)) was obtainedassuming a 1:1 interaction of TFPI and the Fab variant or Fab-Fabconjugate of interest.

Example 20: Effect of Anti-TFPI Antibodies on Tissue Factor-InducedThrombin Generation in Human Haemophilia A Plasma

A synergistic effect of anti-TFPI antibodies on tissue factor-inducedthrombin generation in human haemophilia A plasma can be obtained bycombination or fusion of an antibody targeting an epitope in the KPI-3region with an antibody targeting an epitope in the KPI-1/KPI-2 regionof TFPI.

The effect of antibodies on thrombin generation was studied in athrombin generation assay according to Example 18.

FIG. 1 (Curve a) shows the thrombin generation curve with NHP. Additionof 100 μg/ml sheep anti human FVIII antibody to NHP to simulatehaemophilia A-like condition (Curve b) strongly reduced thrombingeneration. NHP contains about 1.6 nM TFPI of which only about 0.2 nM ispresent as full length TFPIα. Addition of a number of anti-TFPIantibodies, including mAb 2021 (also disclosed in WO2010/072691), toFVIII-neutralised plasma efficiently re-establishes a close to normalthrombin generation curve. However, pre-clinical and clinical studieshave shown that in vivo targeting of TFPI with antibodies or aptamerscauses a significant elevation of the TFPI plasma level. It wastherefore of interest to study the effect of TFPI targeting underconditions with an elevated TFPI concentration. FIG. 1 shows thataddition of 20 nM full-length TFPIα to FVIII-neutralised plasmacompletely prevented a measurable thrombin generation (Curve c) and thataddition of 200 nM of a high affinity antibody against KPI-2 (mAb 2021)could not efficiently abrogate TFPI inhibition and establish a robustthrombin generation under these conditions (Curve d). Addition of 200 nMof a high affinity antibody against KPI-3 (mAb 4F1 10) is completelyincapable of establishing a significant thrombin generation in thepresence of 20 nM TFPI (Curve e). Surprisingly, however, the combinationof 100 nM mAb 2021 and 100 nM mAb 4F110 shows a synergistic effect byestablishing a robust thrombin generation (Curve f) that is clearlylarger than the sum of thrombin generation produced by each antibodyalone and is of the same order of magnitude as the thrombin generationproduced in NHP (Curve a).

The data in FIG. 1 shows that a combination of an antibody against KPI-3of TFPI in combination with an antibody against KPI-2 actedsynergistically in reversing TFPI inhibition at elevated TFPIconcentrations. Parameters of thrombin peak height (Peak) derived fromthe curves in FIG. 1 are listed in Table 1.

TABLE 1 Effect of combinations of anti-TFPI KPI-2 and anti-TFPI KPI-3monospecific antibodies on thrombin generation in FVIII-neutralised NHP(HAP) with 20 nM full-length TFPIα Combination Peak mAb-1 mAb-2 of mAb1and 100 nM 100 nM mAb-2 (nM) NHP¹⁾ — — — 36.0 HAP²⁾ — — — 8.0 HAP +TFPI³⁾ — — — 0 HAP + TFPI³⁾ 2021 2021 2 × KPI-2 18.9 HAP + TFPI³⁾ 20214F110 KPI-2 + KPI-3 62.4 HAP + TFPI³⁾ 4F110 4F110 2 × KPI-3 0 ¹⁾NHP:Normal human plasma without further additions, ²⁾HAP: FVIII-neutralisedplasma, ³⁾HAP + TFPI: FVIII-neutralised plasma with 20 nM TFPIα

A number of antibodies against the C-terminal region of TFPI weresubsequently evaluated for a possible synergistic effect on thrombingeneration, when combined with mAb 2021. Under conditions with anelevated TFPI concentration obtained by addition of 20 nM human TFPIα toFVIII-neutralized plasma, none of the C-terminal antibodies were bythemselves capable of preventing inhibition by TFPI and promoting ameasurable thrombin generation (data not shown). However, the data inTable 2 shows that a number of anti-TFPI KPI-3/C-terminal antibodies(marked *) act synergistically together with mAb 2021 to produce aprominent thrombin peak in plasma under haemophilia A-like conditionswith elevated TFPI concentrations. Thrombin peak height derived fromthrombin generation curves are listed in Table 2.

TABLE 2 Effect of combinations of anti-TFPI KPI-2 and anti-TFPI KPI-3monospecific TFPI antibodies on thrombin generation in FVIII-neutralisedNHP (HAP) with 20 nM full-length TFPIα Ab-1 mAb-2 Combination Peak 100nM 100 nM of Ab targets (nM) NHP¹⁾ — — — 87.1 HAP²⁾ — — — 10.8 HAP +TFPI³⁾ — — — 0 HAP + TFPI³⁾ 2021 2021 2 × KPI-2 17.2 HAP + TFPI³⁾ 20214F110* KPI-2 + KPI-3 50.4 HAP + TFPI³⁾ 2021 22F66  KPI-2 + KPI-3 14.7HAP + TFPI³⁾ 2021 22F71* KPI-2 + KPI-3 26.4 HAP + TFPI³⁾ 2021 22F74*KPI-2 + KPI-3 50.1 HAP + TFPI³⁾ 2021 22F79* KPI-2 + KPI-3 55.6 HAP +TFPI³⁾ 2021  22F132* KPI-2 + KPI-3 64.8 HAP + TFPI³⁾ 2021 35F2* KPI-2 +KPI-3 21.8 HAP + TFPI³⁾ 2021 37F7* KPI-2 + KPI-3 30.6 HAP + TFPI³⁾ 202141F15  KPI-2 + KPI-3 16.4 HAP + TFPI³⁾ 2021 41F30* KPI-2 + KPI-3 27.5HAP + TFPI³⁾ 2021 41F41* KPI-2 + KPI-3 68.3 HAP + TFPI³⁾ 2021 41F61*KPI-2 + KPI-3 38.9 HAP + TFPI³⁾ 2021 41F62  KPI-2 + KPI-3 16.6 HAP +TFPI³⁾ 2021 41F66* KPI-2 + KPI-3 28.7 HAP + TFPI³⁾ 2021 41F74  KPI-2 +KPI-3 17.2 ¹⁾NHP: Normal human plasma without further additions, ²⁾HAP:FVIII-neutralised plasma, ³⁾HAP + TFPI: FVIII-neutralised plasma with 20nM TFPIα, *Synergic effect when combined with mAb 2021

A number of antibodies against the KPI-1 domain of TFPI were evaluatedfor a possible synergistic effect on thrombin generation when combinedwith the KPI-3 domain specific mAb 4F110. Table 3 and Table 4 show dataon the effect of KPI-1 domain specific mAbs 4903 (American Diagnostica,cat no. ADG4903), 1F91, 2F3, 2F22, 2F35, and 2F45 on lag time, time topeak and peak, alone and in combination with mAb 4F110 inFVIII-neutralized plasma (HAP) with 20 nM of human TFPIα added.

Only some of the KPI-1 specific antibodies were by themselves capable ofpreventing inhibition by TFPI and promoting a measurable thrombingeneration. However, the data in Table 4 show that a number of KPI-1domain specific antibodies (marked *) act synergistically together withmAb 4F110 to produce a prominent thrombin peak in plasma underhaemophilia A-like conditions with elevated TFPI concentrations.Thrombin peak height was derived from thrombin generation curves.

TABLE 3 Effect of combinations of anti-TFPI KPI-1 and anti-TFPI KPI-3monospecific TFPI antibodies on thrombin generation in FVIII-neutralisedNHP (HAP) with 20 nM full-length TFPIα mAb-1 mAb-2 Combination Peak 100nM 100 nM of Ab (nM) NHP¹⁾ — — — 36.0 HAP²⁾ — — — 8.0 HAP + TFPI — — — 0HAP + TFPI 4903 4903 2 × KPI-1 0 HAP + TFPI 4903 4F110 KPI-1 + KPI-326.9 ¹⁾NHP: Normal human plasma without further additions, ²⁾HAP:FVIII-neutralised plasma

TABLE 4 Effect of combinations of anti-TFPI KPI-1 and anti-TFPI KPI-3monospecific antibodies on thrombin generation in FVIII-neutralised NHP(HAP) with 20 nM full-length TFPIα mAb-1 mAb-2 Combination Lag timettPeak Peak 100 nM 100 nM of Ab targets (min) (min) (nM) NHP¹⁾ — — — 6.711.6 71.0 HAP + TFPI²⁾ — — — 0 0 0 HAP + TFPI²⁾ 4F110 4F110 2 × KPI-3 00 0 HAP + TFPI²⁾ 1F91 1F91 2 × KPI-1 0 0 0 HAP + TFPI²⁾ 1F91* 4F110KPI-1 + KPI-3 3.1 7.4 18.9 HAP + TFPI²⁾ 2F3 2F3 2 × KPI-1 5.8 14.6 20.9HAP + TFPI²⁾ 2F3* 4F110 KPI-1 + KPI-3 3.3 8.9 63.3 HAP + TFPI²⁾ 2F222F22 2 × KPI-1 5.1 13.6 23.7 HAP + TFPI²⁾ 2F22* 4F110 KPI-1 + KPI-3 3.39.1 61.7 HAP + TFPI²⁾ 2F35 2F35 2 × KPI-1 0 0 0 HAP + TFPI²⁾ 2F35 4F110KPI-1 + KPI-3 0 0 0 HAP + TFPI²⁾ 2F45 2F45 2 × KPI-1 9 23.6 7.2 HAP +TFPI²⁾ 2F45* 4F110 KPI-1 + KPI-3 3.2 10.7 28.5 ¹⁾NHP: Normal humanplasma without further additions, ²⁾HAP: FVIII-neutralised plasma,*Synergic effect on thrombin generation when combined with mAb 4F110

Example 21: Synergistic Effect of Anti-TFPI Fab Fragment Conjugates onTissue Factor-Induced Thrombin Generation in Human Haemophilia a Plasma

Fab fragments and Fab-Fab conjugates prepared according to Examples 6, 710, 12, 13 and 17 were analysed in a binding interaction analysis assayaccording to Example 19 to determine binding affinity towards humanTFPI.

Table 5 shows the equilibrium dissociation constants (K_(D)) for fiveFab fragments:

Fab 0088 (KPI-2) SEQ ID NO: 3+4,

Fab 0094 (KPI-2) SEQ ID NO: 5+6

Fab 0095 (KPI-2) SEQ ID NO: 7+8

Fab 0313 (KPI-2) SEQ ID NO: 58+59

Fab 0089 (KPI-3) SEQ ID NO: 9+10

and four KPI-2/KPI-3 domain specific Fab-Fab conjugates (SEQ ID NOs forthe individual Fabs are the same as above):

Fab 0088-Fab 0089 (also referred to as 9002; KPI-2/KPI-3)

Fab 0094-Fab 0089 (also referred to as 9004; KPI-2/KPI-3)

Fab 0095-Fab 0089 (also referred to as 9005; KPI-2/KPI-3)

Fab 0313-Fab 0089 (also referred to as 9031; KPI-2/KPI-3)

TABLE 5 Binding of anti-TFPI KPI-2 and anti-TFPI KPI-3 Fab fragments andFab-Fab conjugates to human TFPIα (TFPIα) Affinity Ligand Target k_(a)(1/MS) k_(d) (1/s) K_(D) (M) Fab 0088) TFPIα 1.7E+06 1.0E−04 6.0E−11 Fab0089 TFPIα 5.0E+06 5.7E−04 1.1E−10 Fab 0094 TFPIα 1.5E+05 6.6E−044.4E−09 Fab 0095 TFPIα 3.9E+06 3.0E−03 7.8E−10 Fab 0313 TFPIα 4.9E+042.1E−03 4.2E−08 Fab 0088-Fab 0089 (9002) TFPIα 7.1E+06 3.8E−05 5.3E−12Fab 0094-Fab 0089 (9004) TFPIα 6.2E+06 2.2E−04 3.6E−11 Fab 0095-Fab 0089(9005) TFPIα 6.1E+06 2.3E−04 3.8E−11 Fab 0313-FAb 0089 (9031) TFPIα4.0E+07 6.7E−03 1.7E−10

Fab fragments against the KPI-2 domain of TFPI were evaluated for apossible effect on thrombin generation (as measured according to Example18). Table 6 shows data on the effect of KPI-2 domain specific Fabfragments 0088, 0094 and 0095 and mAb 2021 on thrombin peak inFVIII-neutralized NHP (HAP) with endogenous TFPI levels. Fab fragments0088, 0094 and 0095 are variants of mAb 2021. Table 7 shows the resultsfrom a similar experiment wherein 2 nM full-length TFPIα was added toFVIII-neutralized NHP (HAP). Thrombin peak height was derived fromthrombin generation curves.

TABLE 6 Effect of anti-TFPI KPI-2 specific Fab fragments or mAb 2021 onthrombin generation in FVIII-neutralised NHP (HAP) with endogenous TFPIlevels Fab/mAb mAb 2021 Fab 0088 Fab 0094 Fab 0095 conc. (nM) Thrombinpeak (nM) 0.00 22.4 22.4 22.3 25.1 6.25 112.8 96.8 35.9 74.2 12.5 113.698.1 45.0 81.3 25.0 116.5 101.5 60.2 84.9 50.0 118.2 100.9 73.9 85.4 100118.0 87.7 74.1 84.7

TABLE 7 Effect of anti-TFPI KPI-2 specific Fab fragments or mAb 2021 onthrombin generation in FVIII-neutralised NHP (HAP) with 2 nM full-lengthTFPIα Fab/mAb mAb 2021 Fab 0088 Fab 0094 Fab 0095 conc. (nM) Thrombinpeak (nM) 0.00 4.08 4.05 4.1 3.9 6.25 48.9 38.4 5.9 13.3 12.5 61.7 64.210.6 19.4 25.0 68.4 69.8 14.3 22.4 50.0 73.4 67.7 18.9 25.1 100 58.648.1 20.3 24.6

Fab-Fab conjugates targeting the KPI-2 and KPI-3 domains were preparedaccording to examples 6, 7 10 (9002) and 12 (9004) and compared tocorresponding non-conjugated Fab fragments in order to determine apossible effect on thrombin generation as measured according to Example18. Tables 8A and 8B show data on the effect of KPI-2+KPI-3 domainspecific Fab 0088-Fab 0089 (designated 9002) and Fab 0094-Fab 0089(designated 9004) conjugates as well as the Fab fragments in anon-conjugated form on thrombin generation in FVIII-neutralized NHP(HAP) with 20 nM full length TFPIα.

It clearly follows that the Fab-Fab conjugates, in addition to thesynergistic effect obtained by combining to antibodies or Fab fragments,show an avidity effect on thrombin generation when compared to acombination—i.e. non-conjugated—Fab fragments at the same concentration.Thrombin peak height was derived from thrombin generation curves.

TABLE 8A Effect of anti-TFPI KPI-2 and anti-TFPI KPI-3 specific Fabfragments or Fab-Fab conjugates on thrombin generation inFVIII-neutralised NHP (HAP) with 20 nM TFPIα Fab 0088 − Fab 0094 − Fab0089 Fab 0088 + Fab 0089 Fab 0094 + (9002) Fab 0089 (9004) Fab 0089Thrombin peak (nM) NHP 47.2 47.2 47.2 47.2 HA Plasma 10.3 10.3 10.3 10.3HA Plasma + 20 nM TFPI 0 0 0 0 +20 nM Fab-Fab or 22.3 0 2.0 0 Fab + Fab*+50 nM Fab-Fab or 39.7 18.1 14.8 1.3 Fab + Fab** +100 nM Fab-Fab or 46.836.3 19.3 5.1 Fab + Fab*** +200 nM Fab-Fab or 48.2 47.4 24.9 14.1 Fab +Fab***** 2 × 20*, 2 × 50**, 2 × 100*** or 2 × 200**** nM of each of thenon-conjugated Fab fragments was used to achieve a total concentrationcomparable to the concentration of the Fab-Fab conjugates

TABLE 8B Effect of anti-TFPI KPI-2 and anti-TFPI KPI-3 specific Fabfragments or Fab-Fab conjugates on thrombin generation (time to peak) inFVIII-neutralised NHP (HAP) with 20 nM TFPIα Fab 0088 − Fab 0094 − Fab0089 Fab 0089 (Fab-Fab Fab 0088 + (Fab-Fab Fab 0094 + 9002) Fab 00899004) Fab 0089 Time to peak (min) NHP 9.7 9.7 9.7 9.7 HA Plasma 12.712.7 12.7 12.7 HA Plasma + 20 nM TFPI — — — — +20 nM Fab-Fab or 20.4 —~40 — Fab + Fab* +50 nM Fab-Fab or 18.1 23.7 25 ~60 Fab + Fab** +100 nMFab-Fab or 17.5 20.7 17.5 ~45 Fab + Fab*** +200 nM Fab-Fab or 16.4 18.716.0 23.1 Fab + Fab***** 2 × 20*, 2 × 50**, 2 × 100*** or 2 × 200**** nMof each of the non-conjugates Fab fragments was used to achieve a totalconcentration comparable to the concentration of the Fab-Fab conjugates

It clearly follows that the Fab-Fab conjugates show an avidity effect onbinding to TFPI, resulting in a synergistic increase in thrombingeneration which is higher than the effect obtained by combining twoantibodies or two non-conjugated Fab fragments.

Fab fragments and Fab-Fab conjugates targeting the KPI-1 and KPI-3domains were prepared according to Example 6, 7 and 11 (9028) and wereanalysed in a binding interaction analysis assay according to Example 19to determine binding affinity towards human TFPI. Table 9 shows theequilibrium dissociation constants (K_(D)) for one Fab fragment:

Fab 2F22 (KPI-1) (SEQ ID NO: 40 and 41)

and one KPI-1/KPI-3 domain specific Fab-Fab conjugate (SEQ ID NOs forthe individual Fabs are the same as above):

Fab 2F22-Fab 0089 (also referred to as 9028; KPI-1/KPI-3)

TABLE 9 Binding of anti-TFPI KPI-1 Fab fragment 2F22 and of an anti-TFPIKPI-1 and anti-TFPI KPI-3 Fab-Fab conjugate to human TFPIα (TFPIα).Affinity Ligand Target k_(a) (1/MS) k_(d) (1/s) K_(D) (M) Fab 2F22 TFPIα1.2E+05 2.1E−04 1.7E−09 Fab 2F22-Fab 0089 (9028) TFPIα 2.7E+06 2.0E−047.5E−11

Fab-Fab conjugates targeting the KPI-1 and KPI-3 domains were comparedto corresponding non-conjugated Fab fragments in order to determine apossible effect on thrombin generation measured according to example 18.Table 10 shows data on the effect of KPI-1/KPI-3 domain specific Fab2F22-Fab 0089 conjugates as well as the Fab fragments in non-conjugatesform on thrombin generation in FVIII-neutralized NHP (HAP) with 20 nMfull length TFPIα.

TABLE 10 Effect of anti-TFPI KPI-1 and anti-TFPI KPI-3 specific Fabfragments and Fab-Fab conjugate compounds on thrombin generation inFVIII neutralised NHP (HAP) with 20 nM full-length TFPIα FVIIIneutralized Peak (nM) Lag time (min) ttPeak (min) NHP (HAP) Fab Fab FabFab Fab Fab TFPI Drug 2F22 + 2F22 − 2F22 + 2F22 − 2F22 + 2F22 − conc.conc. Fab Fab Fab Fab 0089 Fab Fab Fab Fab 0089 Fab Fab Fab Fab 0089(nM) (nM) 2F22 0089 0089 conjugate 2F22 0089 0089 conjugate 2F22 00890089 conjugate 20 200 26.6 0.97 68.5 87.0 7.7 16.8 4.0 4.0 14.7 76.711.7 10.8 20 100 26.7 0.8 61.7 76.4 8.0 20.3 4.0 4.2 16.3 71.8 12.5 11.020 50 21.2 0.0 54.5 54.1 8.17 n.a. 4.2 4.3 19.8 n.a. 13.0 12.0 20 2511.9 0.0 31.1 21.4 12.0 n.a. 4.7 5.2 29.0 n.a. 15.8 16.2 20 0 0.0 0.00.0 0.0 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. NHP Peak (nM) Lag time(min) ttPeak (min) 61.1 8.3 13.8 ‘Peak’: thrombin peak height; ‘ttPeak’:time to peak. n.a.: not available.

Example 22: Crystal Structure of Soluble TFPI KPI-1 in Complex with FabFragment of Anti-TFPI mAb 2F22

The 3D structure of soluble human TFPI Kunitz-type protease inhibitordomain 1 (KPI-1), inclusive the N-terminal part preceding the TFPIKPI-1, (TFPI 1-79, numbering according to SEQ ID NO: 1), in complex witha Fab fragment (SEQ ID NOs: SEQ 52 and 53) of the anti-TFPI2F22monoclonal antibody, was determined to high resolution using X-raycrystallography. The results demonstrate that the antibody is capable ofbinding the KPI-1 of TFPI, and part of the preceding N-terminal. Theresulting human TFPI epitope residues comprise: Leu 16, Pro 17, Leu 19,Lys 20, Leu 21, Met 22, Phe 25, Cys 35, Ala 37, Met 39, Arg 41, Tyr 56,Gly 57, Gly 58, Cys 59, Glu 60, Gly 61, Asn 62, Gln 63, Arg 65, Phe 66,Glu 67, Glu 71 and Met 75 (SEQ ID NO: 1).

Materials and Methods

To obtain Fab fragments suitable for crystallography, a pTT-basedexpression vector for expression of a truncated mAb 2F22 HC wasgenerated. The VH fragment was excised from a mAb 2F22 HC expressionvector by restriction enzyme digestion and cloned into a linearizedpTT-based toolbox vector containing the sequence for a truncated humanIgG4 constant region. The IgG4-based HCs was truncated in the hingeregion after the lysine corresponding to position 222 in the sequencefor the extended Fab 2F22 HC (Fab for chemical coupling, SEQ ID NO: 40).The cloning reaction was subsequently transformed into E. coli forselection. The sequence of the final construct was verified by DNAsequencing. The Fab fragment of mAb 2F22 fragment was expressed as Fab0296 (SEQ ID NO: 52 and 53) in EXPI293F cells and purified by standardaffinity chromatography using KappaSelect resin as described in example7.

Soluble human TFPI KPI-1, including the N-terminal part of human TFPIand, additionally, a GSSGSSG tag N-terminally attached (SEQ ID NO: 62)and Fab 0296 (which consists of a light chain corresponding to SEQ IDNO: 53 and a heavy chain fragment corresponding to SEQ ID NO: 52), bothin phosphate buffered saline (PBS) buffer (4 tablets in 2 litres ofwater, GIBCO Cat. No. 18912-014 Invitrogen Corporation), were mixed witha slight molar excess (1.1:1) of the TFPI specie. The complex was thenconcentrated to about 10.0 mg/ml using an Amicon Ultra-4 centrifugalfilter with a 10,000 molecular weight cut-off. Crystals were grown bythe sitting drop-technique using a 96 wells TTP IQ plate from TTP LabTech no:4150-05800 and 100 μl precipitant solution per well. Theprecipitant solution contained 20% w/v PEG 3350, 200 mM potassiumformate and was mixed with the protein solution in a ratio of 3:1. Totaldrop size was 200 nl and crystals appeared after a few days. A crystalwas prepared for cryo-freezing by transferring 1 μl of a cryo-solutionmix containing 75% of the precipitant solution and 25% glycerol to thedrop containing the crystal. The soaking was allowed for about 2minutes. The crystal was then fished, flash frozen in liquid N₂ and keptat a temperature of 100 K by a cryogenic N₂ gas stream during datacollection. Crystallographic data were collected, to 1.65 Å at beam-lineBL911-3 at MAX-lab, Lund, Sweden. Space group determination, integrationand scaling of the data were made by the XDS software package [Kabsch,W., J. Appl. Crystallogr., (1993), Vol. 26, pages 795-800]. The spacegroup was determined to be C2 and the cell parameters for thesynchrotron data were determined to be 89.010, 66.660, 106.110 Å,respectively, and with a βangle of 111.18°. The R-sym to 1.65 Åresolution was 8.4% and completeness 99.5%. Mean ofintensity/sigma(intensity) of unique reflections were equal to 2.0 ataround 1.8 Å resolution.

Molecular replacement (MR) was used for structure determination usingthe coordinates of a Fab molecule with accession code 1NGZ [Yin, J. etal, Proc Natl Acad Sci USA. 2003, Feb. 4, (100), Vol. 100 pages 856-861]of the Protein Data Bank (PDB) [Berman, H. M. et al, Nucleic Acids Res.,(2000), Vol. 28, pages 235-242]. The Fab molecule was divided into twodomains, the variable and the constant domains, which each were used assearch models in the MR calculations. The Molrep software [Vagin, A. etal, J. Appl. Crystallogr., (1997), Vol. 30, pages 1022-1025] of the CCP4package CCP4 [Collaborative Computational Project, N., Actacrystallographica. Section D, Biological crystallography, (1994), Vol.50, pages 760-763] was used to find the positions of the constant andvariable Fab domains. The KPI-1 domain was not found in the MR step,however, the difference electron density map indicated the approximatepositions of the KPI-1 domain molecules at this stage. After electrondensity improvements by the DM software of the CCP4 software package,followed by automated model building and phase improvements using theARP-wARP software [Langer, G. et al, Nat Protoc, (2008), Vol. 3, pages1171-1179][Murshudov, G. N. et al, Acta Crystallographica Section DBiological Crystallography, (2011), Vol. 67, pages 355-367] gave analmost complete structure of both the Fab 0296 molecule and of the KPI-1domain structure, and part of the N-terminal preceding the KPI-1 domain.For the TFPI (SEQ ID NO: 1) residues from 15 to 77 are included in theX-ray model which in addition to the KPI-1 also includes some residuesN-terminally of the KPI-1 (residues 26-79). For the Fab 0296 fragmentthe light chain residues 1 to 212 and the heavy chain residues 1 to 221are observed. A procedure of computer graphics inspection of theelectron density maps, model corrections and building using the Cootsoftware program [Emsley, P. et al, Acta Crystallogr. Sect. D-Biol.Crystallogr., (2004), Vol. 60, pages 2126-2132] followed bycrystallographic refinements, using the software programs Refmac5[Murshudov, G. N. et al, Acta Crystallographica Section D BiologicalCrystallography, (2011), Vol. 67, pages 355-367] of the CCP4 softwarepackage was entered. The procedure was cycled until no furthersignificant improvements could be made to the model. Final R- and R-freefor all data to 1.65 Å resolution were 0.192 and 0.220, respectively.

Results

Calculation of the average areas excluded in pair-wise interactions bythe software program Areaimol [Lee, B. et al, J Mol Biol, (1971), Vol.55, pages 379-400][Saff, E. B. et al, Math Intell, (1997), Vol. 19,pages 5-11] of the CCP4 program suite [Collaborative ComputationalProject, N., Acta crystallographica. Section D, Biologicalcrystallography, (1994), Vol. 50, pages 760-763] gave for the solublehuman TFPI fragment/anti-TFPI Fab 0296 molecular complex of the crystalstructure 1195 Å².

The direct contacts between the TFPI KPI-1, inclusive the N-terminalpart of TFPI observed in the crystal structure, (SEQ ID NO:62) andanti-TFPI Fab 0296 (SEQ ID NOs: 52 and 53) were identified by runningthe Contacts software of the CCP4 program suite [Bailey, S., ActaCrystallogr. Sect. D-Biol. Crystallogr., (1994), Vol. 50, pages 760-763]using a cut-off distance of 4.0 Å between the anti-TFPI Fab 0296 and theTFPI fragment molecules. The results from the soluble TFPIfragment/anti-TFPI Fab 0296 complex crystal structure are shown in Table11. The resulting TFPI KPI-1, including the TFPI N-terminal, epitope foranti-TFPI2F22 was found to comprise one or more of the followingresidues of TFPI (SEQ ID NO: 1): Leu 16, Pro 17, Leu 19, Lys 20, Leu 21,Met 22, Phe 25, Cys 35, Ala 37, Met 39, Arg 41, Tyr 56, Gly 57, Gly 58,Cys 59, Glu 60, Gly 61, Asn 62, Gln 63, Arg 65, Phe 66, Glu 67, Glu 71and Met 75. Evaluated from distances, charge-charge interactions,hydrogen bonds, polar and hydrophobic interactions and low solventaccessibility the following residues seems to be particularly importantresidues of the epitope: Arg 41, Arg 65 and Glu 67.

Thus, the anti-TFPI2F22 TFPI epitope comprises residues preceding theKPI-1, including a short N-terminal α-helix, residues in the loop beforeβ-strands 1 of the KPI-1 and residues in the beginning of α-strand 1. Italso includes residues in the end of β-strand 2 and residues in the loopbetween β-strand 2 and the C-terminal α-helix of KPI-1 and residueswithin the C-terminal α-helix of KPI-1.

The anti-TFPI2F22 paratope for TFPI KPI-1 includes residues Val 2, Phe27, Tyr 32, Trp 52, Arg 53, Gly 54, Gly 55, Ser 56, Ile 57, Asp 58, Tyr59, Ala 61, Met 64, Lys 97, Ser 99, His 100, Asn 102, Tyr 103, Val 104,Gly 105 and Tyr 106 of the heavy (H) chain (SEQ ID NO:52, Table 11), andresidues Pro 31, Ala 32, Tyr 49, Ser 50, Asn 53, Tyr 55, Thr 56, Tyr 91,Thr 92, Ser 93 and Tyr 94 of the light (L) chain (SEQ ID NO: 53, Table11).

Table 11. Interactions

TFPI KPI-1, chain K, (SEQ ID NO: 1) interactions with the heavy chain(chain H) of anti-TFPI Fab 0296 (SEQ ID NO: 52) and light chain (chainL) of anti-TFPI Fab 0296 (SEQ ID NO: 53) for the first crystallographicindependent complex. A distance cut-off of 4.0 Å was used. The contactswere identified by the CONTACT computer software program of the CCP4suite [Collaborative Computational Project, N., Acta crystallographica.Section D, Biological crystallography, (1994), Vol. 50, pages 760-763].In the last column “***” indicates a strong possibility for a hydrogenbond at this contact (distance <3.3 Å) as calculated by CONTACT, “*”indicates a weak possibility (distance >3.3 Å). Blank indicates that theprogram considered there to be no possibility of a hydrogen bond.Hydrogen-bonds are specific between a donor and an acceptor, aretypically strong, and are easily identifiable.

TABLE 11 Interactions These results indicate that anti-TFPI Fab 0296specifically binds to TFPI KPI-1 and part of the preceding N-terminal.hTFPI Fab 0296 Res. # Res. # Res. and Atom Res. and Atom DistancePossibly Type Chain name Type Chain name [Å] H-bond Leu 16K CB Tyr 32HOH 3.77 Leu 16K CD1 Phe 27H CB 3.99 Tyr 32H CZ 3.78 Tyr 32H CE2 3.86 Tyr32H OH 3.40 Lys 97H CE 3.77 Leu 16K CD2 Val 2H CG2 3.74 Pro 17K CG Thr56L OG1 3.68 Leu 19K CA Tyr 49L OH 3.80 Leu 19K CB His 100H ND1 3.95 His100H CE1 3.46 His 100H NE2 3.73 Tyr 49L OH 3.79 Leu 19K CD1 Ser 99H O3.47 His 100H CD2 3.85 His 100H NE2 3.69 Tyr 55L CE1 3.86 Leu 19K C Tyr49L OH 3.85 Lys 20K N Tyr 49L CZ 3.89 Tyr 49L OH 3.01 *** Lys 20K CA Tyr49L OH 3.93 Lys 20K CB Tyr 49L OH 3.70 Lys 20K CG Tyr 49L OH 3.51 Lys20K CD Tyr 49L OH 3.80 Asn 53L CG 3.73 Asn 53L OD1 3.29 Lys 20K CE Asn53L OD1 3.93 Lys 20K C His 100H CE1 3.92 Lys 20K O His 100H ND1 3.41 *His 100H CE1 2.88 Tyr 49L CE2 3.58 Leu 21K CA Tyr 106H OH 3.73 Leu 21KCB Tyr 106H OH 3.99 Leu 21K CD2 Tyr 103H CE2 3.72 Tyr 103H CD2 3.52 His100H ND1 3.89 Tyr 106H OH 3.87 Leu 21K C Tyr 106H OH 3.71 Met 22K N Tyr106H CZ 3.81 Tyr 106H OH 2.81 *** Tyr 106H CE2 3.87 Met 22K CA Tyr 106HOH 3.68 Met 22K CB Tyr 106H OH 3.60 Met 22K CG Ser 50L OG 3.75 Met 22KSD Pro 31L CG 3.78 Met 22K CE Pro 31L CG 3.80 Met 22K O Tyr 106H OH3.86 * Phe 25K CZ Gly 105H O 3.58 Ala 32L CB 3.89 Phe 25K CE2 Val 104H O3.96 Gly 105H CA 3.87 Gly 105H C 3.87 Gly 105H O 3.41 Tyr 106H CE1 3.90Tyr 106H CZ 3.61 Tyr 106H OH 3.79 Tyr 106H CE2 3.86 Phe 25K CD2 Val 104HO 3.81 Tyr 106H CZ 3.81 Tyr 106H OH 3.59 Cys 35K SG Ala 61H CB 3.72 Ala37K CB Met 64H CE 3.75 Met 39K SD Ile 57H O 3.21 Met 39K CE Ser 56H CA3.87 Ser 56H CB 3.66 Ile 57H N 3.44 Ile 57H O 3.31 Arg 41K NE Ser 56H CB3.74 Arg 41K CZ Ser 56H CB 3.85 Asp 58H OD1 3.00 Arg 41K NH1 Asp 58H CG3.53 Asp 58H OD1 2.67 *** Asp 58H OD2 3.65 * Arg 41K NH2 Ser 56H CB 3.50Ile 57H N 3.79 * Ile 57H C 3.58 Ile 57H O 3.12 *** Asp 58H CG 3.78 Asp58H OD1 2.58 *** Tyr 56K CE2 Asp 58H OD1 3.40 Gly 57K O Met 64H CE 3.82Gly 58K O Asp 58H C 3.65 Tyr 59H N 2.83 *** Tyr 59H CB 3.96 Tyr 59H CD13.80 Asp 58H CA 3.56 Tyr 59H CA 3.82 Tyr 59H O 3.69 * Cys 59K CA Tyr 59HO 3.61 Cys 59K CA Tyr 59H O 3.59 Cys 59K CB Tyr 59H O 3.52 Cys 59K CBTyr 59H O 3.30 Cys 59K SG Met 64H SD 3.34 Ala 61H CA 3.60 Tyr 59H O 3.92Ala 61H N 3.94 Ala 61H CB 3.75 Cys 59K SG Ala 61H CB 3.85 Glu 60K N Ser93L O 3.90 * Glu 60K CA Ser 93L O 3.25 Ser 93L CB 4.00 Ser 93L C 3.99Glu 60K OE2 Tyr 94L CE1 3.99 Glu 60K C Ser 93L O 3.30 Glu 60K O Ser 93LO 3.25 *** Gly 61K O Ser 93L CA 3.27 Ser 93L CB 3.51 Asn 62K CA Thr 92LO 3.84 Gln 63K CA Val 104H CG1 3.96 Gln 63K CG Tyr 91L O 3.58 Gly 105HCA 3.77 Gln 63K CD Tyr 91L O 3.77 Thr 92L CA 3.83 Gln 63K OE1 Thr 92L O3.95 * Thr 92L CA 3.87 Gln 63K NE2 Tyr 91L O 3.65 * Thr 92L CA 3.98 Thr92L CG2 3.73 Ala 32L CB 3.74 Arg 65K NE Asp 58H OD1 3.91 * Asp 58H OD23.76 * Arg 65K CZ Asp 58H OD2 3.81 Arg 65K NH2 Asp 58H CG 3.65 Asp 58HOD2 2.92 *** Val 104H CG1 3.71 Val 104H CG2 3.60 Arg 65K O Val 104H CG23.64 Phe 66K CD1 Tyr 103H CE1 3.80 Tyr 103H CD1 3.77 Phe 66K CE1 Tyr103H CE1 3.74 Tyr 103H CD1 3.63 Glu 67K CB Asn 102H ND2 3.46 Glu 67K CGSer 56H OG 3.33 Glu 67K CD Gly 54H N 3.40 Gly 54H CA 3.69 Ser 56H OG3.32 Trp 52H CB 3.78 Asn 102H ND2 3.94 Ser 56H CB 3.89 Glu 67K OE1 Gly54H N 2.79 *** Gly 54H CA 3.49 Trp 52H CB 3.57 Trp 52H C 3.94 Arg 53H N3.47 * Arg 53H CG 3.98 Arg 53H CD 3.87 Arg 53H C 3.84 Asn 102H CG 3.86Asn 102H OD1 3.93 * Asn 102H ND2 2.97 *** Glu 67K OE2 Gly 54H C 3.27 Gly54H O 3.77 * Gly 55H N 3.51 * Ser 56H N 3.02 *** Gly 54H N 3.28 *** Gly54H CA 3.27 Ser 56H CA 3.64 Ser 56H OG 2.52 *** Trp 52H CB 3.91 Ser 56HCB 3.10 Glu 71K CD Asn 102H ND2 3.46 Arg 53H NH1 3.50 Glu 71K OE1 Asn102H CB 3.82 Asn 102H CG 3.79 Asn 102H ND2 2.81 *** Glu 71K OE2 Arg 53HNH2 3.78 * Arg 53H CD 3.65 Arg 53H NE 3.58 * Asn 102H ND2 3.43 * Arg 53HCZ 3.01 Arg 53H NH1 2.28 *** Glu 71K O Tyr 103H CE1 3.91 Tyr 103H OH3.57 * Met 75K N Tyr 103H OH 3.80 * Met 75K CB Tyr 103H CE1 3.82 Tyr103H CZ 4.00 Tyr 103H OH 3.73These results show that anti-TFPI Fab 0296 specifically binds to TFPIKPI-1 and part of the preceding N-terminal.

Example 23: Binding of Antibodies to Cell Surface Associated TFPI

The ability of anti-TFPI antibodies, Fab fragments or Fab-Fab conjugatesto bind to TFPI associated with the endothelial cell surface in vitrowas studied using flow cytometry. Human umbilical vein cell lineEA.hy926 cells (ATCC) were incubated for 30 min on ice with differentconcentrations of anti-TFPI mAb 2021, Fab 0088, Fab 0094, Fab 0095, orFab 0313, or Fab 0088-Fab 0089 conjugate 9002 or Fab 0094-Fab 0089conjugate 9004 in assay buffer (PBS supplemented with 2% FCS). The cellswere washed with PBS and subsequently incubated on ice withAPC-conjugated secondary antibody (Allophycocyanin-AffiniPure F(ab′)2Fragment Donkey Anti-Human IgG (H+L), Jackson ImmunoResearch, Cat.709-136-149) for 30 min in assay buffer. The cells were washed with PBSand analysed on a BD LSRFortessa flow cytometer. Binding profiles wereplotted using the median fluorescence intensity at different antibodyconcentrations and half maximal binding values (EC50) were calculated.

The data listed in Table 12 clearly shows that KPI-2 TFPI Fab 0088, Fab0094, and Fab 0313 (all Fab fragments are derived from mAb 2021) bindwith similar affinities to EA.hy926 cell surface associated TFPI andTFPIα as determined by SPR analysis listed in Table 5. In contrast tohigh affinity binding to TFPIα (Table 5), no measurable binding ofanti-TFPI KPI-3 mAb 4F110 could be detected to EA.hy926 cell surfaceassociated TFPI. The data also shows that the affinity of Fab 0094-Fab0089 conjugate 9004 for EA.hy926 cell surface associated TFPI issignificantly reduced compared to its affinity for TFPIα (Table 5)indicating preferential binding to TFPI species which comprises aC-terminal region.

TABLE 12 EC50 values for the binding of anti-TFPI antibodies andfragments EA.hy926 cell surface associated TFPI EC50 Antibody(mAb/Fab/Fab-Fab conjugate) (nM) mAb 2021 0.11 Fab 0088 0.31 Fab 00943.80 Fab 0313 25.90 mAb 4F110 n.b. Fab 0088-Fab 0089conjugate (9002)0.33 Fab 0094-Fab 0089 conjugate (9004) 6.90 (n.b: No measurablebinding)

Example 24: Neutralisation by mAbs and Fab-Fab Conjugates of the TFPIβInhibition of TF/FVIIa-Mediated FXa Generation

Human umbilical vein cell line EA.hy926 cells (ATCC) were grown to 100%confluence in 96 well plates in DMEM (Gibco) supplied with 10% FCS and1% penicillin/streptomycin. The cells were stimulated with 20 ng/ml TNFαand 20 ng/ml IL-1β for 3 hours and used for testing of neutralisation ofcell surface TF-dependent FX activation. Cells were washed twice with 25mM HEPES, 137 mM NaCl, 3.5 mM KCl, pH 7.4 before FX activation wasmeasured in 25 mM HEPES, 137 mM NaCl, 3.5 mM KCl, 5 mM CaCl₂, 1 mg/mlBSA pH 7.4. After incubation for 15 min at 37° C., neutralisation ofTFPI was induced by addition of 0-60 nM mAb or Fab-Fab conjugate for 120min. This was followed by incubation with 50 pM rFVIIa (Novo NordiskA/S) for 15 min. Generation of FXa was then initiated by addition of 50nM FX for 40 min at 37° C. The reaction was stopped by mixing 40 μlsupernatant with 10 μl (75 mM) EDTA and FXa activity was finallymeasured with 0.6 mM chromogenic substrate S-2765 (Chromogenix).TF/FVIIa activity was verified in control experiments with addition of0.5 mg/ml neutralising goat anti human TF polyclonal antibody.

Data were analysed with GraphPad Prism Software (version 5). Results areshown in FIG. 2. FXa activity in the supernatant was measured afterincubation with 50 pM FVIIa and 50 nM FX at 37° C. with variousconcentrations (0-60 nM) of mAb 2021: (A); mAb 4F110 (B); Fab 0088-Fab0089 conjugate 9002 (C); or Fab 0094-Fab 0089 conjugate 9004 (D).Squares show the effect of 0.5 mg/ml goat anti human TF polyclonalantibody together with 60 nM mAb or Fab-Fab conjugate. Results are shownas mean±SD, n=3.

The data show that mAb 2021, but not mAb 4F110, was capable ofneutralizing TFPI inhibition of TF/FVIIa-mediated FX activation on thesurface of EA.hy926 cells. Neutralization of TFPIβ was also observedwith Fab 0088-Fab 0089 conjugate 9002. In contrast, Fab 0094-Fab 00899004 was without a significant neutralizing effect on the TFPIβinhibition of TF/FVIIa on EA.hy926 cells. Modulation of the affinity ofa KPI-2 binding antibody is therefore shown to be crucial for thefunctional effect Fab-Fab conjugates against KPI-2 and KPI-3.

Example 25: Generation of Asymmetric Bispecific Antibodies

A bispecific TFPI KPI-2/KPI-3 binding antibody antibody was generatedbased on an asymmetric IgG format, i.e through FC heterodimerization. Inorder to achieve FC heterodimerization a set of compatible mutationswere engineered into the FC region of hIgG1 variants of mAb 2021 and mAb4F110.

The VH fragments were excised from the mAb 2021 and mAb 4F110 HCexpressions vectors by restriction enzyme digestion and cloned into alinearized pTT-based toolbox vector containing the sequence for a humanIgG1 constant region. The cloning reaction was subsequently transformedinto E. coli for selection. The sequence of the final construct wasverified by DNA sequencing.

To support FC heterodimerization by controlled Fab-arm exchange a singlephenylalanine to leucine mutation was introduced into the CH3 domain ofthe IgG1 HC of mAb 2021, corresponding to position 409 in SEQ ID NO: 63.The mutations was introduced by site-directed mutagenesis using theQuikChange® Site-Directed mutagenesis kit from Stratagene. The sequencesof all final construct were verified by DNA sequencing. The engineeredIgG1 version of mAb 2021 was expressed as mAb 0309 (SEQ ID NO: 63 and64) in EXPI293F cells as described in example 7. Purification wasperformed by standard affinity chromatography using MabSelectSuRe resinfrom GE Healthcare according to manufacturer's instructions. Thepurified antibodies were buffer exchanged to PBS buffer pH7.2.

A matching lysine to arginine mutation was introduced into the CH3domain of the IgG1 HC of mAb 4F110, corresponding to position 410 in SEQID NO: 65. The mutations was introduced by site-directed mutagenesisusing the QuikChange® Site-Directed mutagenesis kit from Stratagene. Thesequences of all final construct were verified by DNA sequencing. Theengineered IgG1 version of mAb 2021 was expressed as mAb 0312 (SEQ IDNO: 65 and 66) in EXPI293F cells as described in example 7. Purificationwas performed by standard affinity chromatography using MabSelectSuReresin from GE Healthcare according to manufacturer's instructions. Thepurified antibodies were buffer exchanged to PBS buffer pH7.2.

A bispecific KPI-2/KPI-3 binding antibody was prepared by controlledFab-arm exchange between the engineered IgG1 variant of mAb2021 (KPI-2binding), mAb309 and the engineered IgG1 variant of mAb4F110 (KPI-3binding), mAb312. The bispecific antibody was prepared essentiallyaccording to published methods (Labrijn et al., PNAS, 110: 5145-5150(2013)).

In short, mAb 0309 (mAb2021 IgG1 Phe-to-Leu variant SEQ ID NO: 63) wasmixed with mAb 0312 (mAb 4F110 IgG1 Lys-to-Arg variant SEQ ID NO: 65) ata 1:1 molar ratio (1 mg/ml per antibody) in PBS buffer. The antibodieswere selectively reduced by incubation of the 1:1 mixture for 90 minutesat 37° C. in the presence of 25 mM 2-mercaptoethylamine (MEA) to formhalf antibodies and allow Fab-arm exchange. Finally, spontaneousreassembly and re-oxidation was obtained after removal of the reducingagent (diafiltration in to PBS buffer without MEA) and storage at +5° C.for at least 16 hours. Hetero-dimerization, promoted by the CH3 domainmutations, was observed with an efficiency exceeding 90%. The finalbispecific antibody derived from mAb 0309 and 0312 was named mAb 0325(SEQ ID NO: 67-70).

The reverse set of mutations, i.e Lys-to-Arg on Mab 2021 IgG1 andPhe-to-Leu on Mab 4F110 IgG1 were also generated and tested with similarresults.

A synergistic effect on tissue factor-induced thrombin generation inhuman haemophilia A plasma can be obtained by using an asymmetricbispecific antibody, assembled through FC heterodimerisation ofantibodies targeting epitopes in the KPI-2 and KPI-3 regions of TFPI.

The effect of antibodies on thrombin generation was studied in athrombin generation assay according to Example 18.

FIG. 3 (Curve a) shows the thrombin generation curve with NHP. Additionof 100 μg/ml sheep anti human FVIII antibody to NHP to simulatehaemophilia A-like condition (Curve b) strongly reduced thrombingeneration. NHP contains about 1.6 nM TFPI of which only about 0.2 nM ispresent as full length TFPIα. Addition of a number of anti-TFPIantibodies, including mAb 0309 an FC engineered version of mAb 2021 (mAb2021 also disclosed in WO2010/072691), to FVIII-neutralised plasmaefficiently re-establishes a close to normal thrombin generation curve(data not shown). However, pre-clinical and clinical studies have shownthat in vivo targeting of TFPI with antibodies or aptamers causes asignificant elevation of the TFPI plasma level. It was therefore ofinterest to study the effect of TFPI targeting under conditions with anelevated TFPI concentration. FIG. 3 shows that addition of 20 nMfull-length TFPIα to FVIII-neutralised plasma completely prevented ameasurable thrombin generation (Curve c) and that addition of 200 nM ofa high affinity antibody against KPI-2 (mAb 0309) could not efficientlyabrogate TFPI inhibition and establish a robust thrombin generationunder these conditions (Curve d). Addition of 200 nM of a high affinityantibody against KPI-3 (mAb 0312, an FC engineered version of mAb 4F110)is completely incapable of establishing a significant thrombingeneration in the presence of 20 nM TFPI (Curve e). In contrast, theasymmetric bispecific antibody combination of mAb 0309 and 0312; mAb0325 shows a synergistic effect by establishing a robust thrombingeneration (Curve f) that is clearly larger than the sum of thrombingeneration produced by each antibody alone and is of the same order ofmagnitude as the thrombin generation produced in NHP (Curve a).

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

The invention claimed is:
 1. A pharmaceutical composition comprising: afirst monospecific Tissue Factor Pathway Inhibitor (TFPI) antibodycapable of specifically binding to a epitope within Kunitz-type proteaseinhibitor (KPI)-2 and a second monospecific TFPI antibody capable ofspecifically binding to a epitope within KPI-3, wherein the heavy chainof the antibody that binds to the KPI-2 epitope comprises: acomplementarity-determining region (CDR)1 sequence corresponding toamino acid residues 31 to 35 of SEQ ID NO: 3; a CDR2 sequencecorresponding to amino acid residues 50 to 66 of a sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:7; a CDR3 sequence corresponding to amino acid residues 99 to 110 of SEQID NO: 3; and wherein the light chain that binds to the KPI-2 epitopecomprises: a CDR1 sequence corresponding to amino acid residues 24 to 39of SEQ ID NO: 4; a CDR2 sequence corresponding to amino acid residues 55to 61 of SEQ ID NO: 4; a CDR3 sequence corresponding to amino acidresidues 94 to 102 of SEQ ID NO: 4; and wherein the heavy chain of theantibody that binds to the KPI-3 epitope comprises: a CDR1 sequencecorresponding to amino acid residues 31 to 35 of SEQ ID NO: 9; a CDR2sequence corresponding to amino acid residues 50 to 66 of SEQ ID NO: 9;a CDR3 sequence corresponding to amino acid residues 99 to 107 of SEQ IDNO: 9; and wherein the light chain of the antibody that binds to theKPI-3 epitope comprises: a CDR1 sequence corresponding to amino acidresidues 24 to 34 of SEQ ID NO: 10; a CDR2 sequence corresponding toamino acid residues 50 to 56 of SEQ ID NO: 10; a CDR3 sequencecorresponding to amino acid residues 89 to 97 of SEQ ID NO:
 10. 2. Apharmaceutical composition comprising: a first monospecific TFPIantibody capable of specifically binding to an epitope within KPI-1 anda second monospecific TFPI antibody capable of specifically binding toan epitope within KPI-3, wherein the heavy chain of the antibody thatbinds to KPI-1 comprises: a CDR1 sequence corresponding to amino acids31 to 36 of SEQ ID NO: 22; a CDR2 sequence corresponding to amino acids51 to 66 of SEQ ID NO: 22; a CDR3 sequence corresponding to amino acids99 to 104 of SEQ ID NO: 22, and wherein the light chain of the antibodythat binds to KPI-1 comprises: a CDR1 sequence corresponding to aminoacids 24 to 33 of SEQ ID NO: 23; a CDR2 sequence corresponding to aminoacids 49 to 55 of SEQ ID NO: 23; a CDR3 sequence corresponding to aminoacids 88 to 96 of SEQ ID NO: 23; wherein the heavy chain of the antibodythat binds to the KPI-3 epitope comprises: a CDR1 sequence correspondingto amino acid residues 31 to 35 of SEQ ID NO: 9; a CDR2 sequencecorresponding to amino acid residues 50 to 66 of SEQ ID NO: 9; a CDR3sequence corresponding to amino acid residues 99 to 107 of SEQ ID NO: 9;and wherein the light chain of the antibody that binds to the KPI-3epitope comprises: a CDR1 sequence corresponding to amino acid residues24 to 34 of SEQ ID NO: 10; a CDR2 sequence corresponding to amino acidresidues 50 to 56 of SEQ ID NO: 10; a CDR3 sequence corresponding toamino acid residues 89 to 97 of SEQ ID NO:
 10. 3. A pharmaceuticalcomposition comprising: a first monospecific TFPI antibody capable ofspecifically binding to a epitope within KPI-1 and a second monospecificTFPI antibody capable of specifically binding to a epitope within KPI-3,wherein the heavy chain of the antibody that binds to KPI-1 comprises: aCDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 24; aCDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 24; aCDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 24;and wherein the light chain of the antibody that binds to KPI-1comprises: a CDR1 sequence corresponding to amino acids 24 to 34 of SEQID NO: 25; a CDR2 sequence corresponding to amino acids 50 to 56 of SEQID NO: 25; a CDR3 sequence corresponding to amino acids 89 to 96 of SEQID NO: 25; and wherein the heavy chain of the antibody that binds to theKPI-3 epitope comprises: a CDR1 sequence corresponding to amino acidresidues 31 to 35 of SEQ ID NO: 9; a CDR2 sequence corresponding toamino acid residues 50 to 66 of SEQ ID NO: 9; a CDR3 sequencecorresponding to amino acid residues 99 to 107 of SEQ ID NO: 9; andwherein the light chain of the antibody that binds to the KPI-3 epitopecomprises: a CDR1 sequence corresponding to amino acid residues 24 to 34of SEQ ID NO: 10; a CDR2 sequence corresponding to amino acid residues50 to 56 of SEQ ID NO: 10; a CDR3 sequence corresponding to amino acidresidues 89 to 97 of SEQ ID NO:
 10. 4. A pharmaceutical compositioncomprising: a first monospecific TFPI antibody capable of specificallybinding to a epitope within KPI-1 and a second monospecific TFPIantibody capable of specifically binding to a epitope within KPI-3,wherein the heavy chain of the antibody that binds to KPI-1 comprises: aCDR1 sequence corresponding to amino acid residues 31 to 35 of SEQ IDNO: 26; a CDR2 sequence corresponding to amino acid residues 50 to 65 ofSEQ ID NO: 26; a CDR3 sequence corresponding to amino acid residues 98to 110 of SEQ ID NO: 26; and wherein the light chain that binds to KPI-1comprises: a CDR1 sequence corresponding to amino acid residues 24 to 34of SEQ ID NO: 27; a CDR2 sequence corresponding to amino acid residues50 to 56 of SEQ ID NO: 27; a CDR3 sequence corresponding to amino acidresidues 89 to 96 of SEQ ID NO: 27; and wherein the heavy chain of theantibody that binds to the KPI-3 epitope comprises: a CDR1 sequencecorresponding to amino acid residues 31 to 35 of SEQ ID NO: 9; a CDR2sequence corresponding to amino acid residues 50 to 66 of SEQ ID NO: 9;a CDR3 sequence corresponding to amino acid residues 99 to 107 of SEQ IDNO: 9; and wherein the light chain of the antibody that binds to theKPI-3 epitope comprises: a CDR1 sequence corresponding to amino acidresidues 24 to 34 of SEQ ID NO: 10; a CDR2 sequence corresponding toamino acid residues 50 to 56 of SEQ ID NO: 10; a CDR3 sequencecorresponding to amino acid residues 89 to 97 of SEQ ID NO:
 10. 5. Apharmaceutical composition comprising: a first monospecific TFPIantibody capable of specifically binding to a epitope within KPI-1 and asecond monospecific TFPI antibody capable of specifically binding to aepitope within KPI-3, wherein the heavy chain of the antibody that bindsto KPI-1 comprises: a CDR1 sequence corresponding to amino acids 31 to35 of SEQ ID NO: 30; a CDR2 sequence corresponding to amino acids 50 to65 of SEQ ID NO: 30; a CDR3 sequence corresponding to amino acids 98 to110 of SEQ ID NO: 30; and wherein the light chain of the antibody thatbinds to KPI-1 comprises: a CDR1 sequence corresponding to amino acids24 to 34 of SEQ ID NO: 31; a CDR2 sequence corresponding to amino acids50 to 56 of SEQ ID NO: 31; a CDR3 sequence corresponding to amino acids89 to 96 of SEQ ID NO: 31; and wherein the heavy chain of the antibodythat binds to the KPI-3 epitope comprises: a CDR1 sequence correspondingto amino acid residues 31 to 35 of SEQ ID NO: 9; a CDR2 sequencecorresponding to amino acid residues 50 to 66 of SEQ ID NO: 9; a CDR3sequence corresponding to amino acid residues 99 to 107 of SEQ ID NO: 9;and wherein the light chain of the antibody that binds to the KPI-3epitope comprises: a CDR1 sequence corresponding to amino acid residues24 to 34 of SEQ ID NO: 10; a CDR2 sequence corresponding to amino acidresidues 50 to 56 of SEQ ID NO: 10; a CDR3 sequence corresponding toamino acid residues 89 to 97 of SEQ ID NO:
 10. 6. A method for treatingcoagulopathy comprising: administering to a patient in need of suchtreatment a therapeutically effective amount of the pharmaceuticalcomposition according to claim
 1. 7. A method for treating coagulopathycomprising: administering to a patient in need of such treatment atherapeutically effective amount of the pharmaceutical compositionaccording to claim
 2. 8. A method for treating coagulopathy comprising:administering to a patient in need of such treatment a therapeuticallyeffective amount of the pharmaceutical composition according to claim 3.9. A method for treating coagulopathy comprising: administering to apatient in need of such treatment a therapeutically effective amount ofthe pharmaceutical composition according to claim
 4. 10. A method fortreating coagulopathy comprising: administering to a patient in need ofsuch treatment a therapeutically effective amount of the pharmaceuticalcomposition according to claim 5.